Combination therapy for use in cancer therapy

ABSTRACT

The present invention provides methods of treating anal or vaginal tumors and cancers, comprising the step of administering to a subject a combination therapy comprising a chemo-radiation therapy and a recombinant  Listeria  strain.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/063,828, filed on Oct. 14, 2014 and U.S. Provisional Application No. 62/065,973, filed on Oct. 20, 2014, both of which are incorporated by reference herein in their entirety.

FIELD OF INVENTION

The present invention provides methods of treating anal or vaginal tumors and anal or vaginal cancers, comprising the step of administering to a subject a combination therapy comprising a chemo-radiation therapy and a recombinant Listeria strain.

BACKGROUND OF THE INVENTION

Listeria monocytogenes (Lm) is a food-borne gram-positive bacterium that can occasionally cause disease in humans, in particular elderly individuals, newborns, pregnant women and immunocompromised individuals. In addition to strongly activating innate immunity and inducing a cytokine response that enhances antigen-presenting cell (APC) function, Lm has the ability to replicate in the cytosol of APCs after escaping from the phagolysosome, mainly through the action of the listeriolysin O (LLO) protein. This unique intracellular life cycle allows antigens secreted by Lm to be processed and presented in the context of both MHC class I and II molecules, resulting in potent cytotoxic CD8⁺ and Th1 CD4⁺ T-cell-mediated immune responses. Lm has been extensively investigated as a vector for cancer immunotherapy in pre-clinical models.

Persistent infection with high-oncogenic risk human papillomavirus (HR-HPV) types is recognized as a necessary, but not sufficient, cause of invasive carcinoma of the cervix (ICC). HPVs 16 and 18 are the most prevalent types in malignant lesions, accounting for over 70% of ICC and over 50% of high-grade precursor lesions.

Anal cancer is a rare malignancy that begins in the anus, which is the opening at the end of the rectum. The American Cancer Society estimates that 7,270 cases of anal cancer will be diagnosed in 2015 (with the incidence still increasing) and about 1,010 deaths will occur that year from anal cancer. Approximately half of all anal cancers are diagnosed before the malignancy has spread beyond the primary site, whereas 13% to 25% are diagnosed after the cancer has spread to the lymph nodes, and 10% are diagnosed after the cancer has spread to distant organs, or has metastasized. When it is found early, anal cancer is highly treatable, however, if the cancer has spread to distant organs, about one in five patients lives for five years or more.

Receptive anal intercourse is strongly related to the development of anal cancer. Anal infection with human papillomavirus (HPV) resulting in genital warts is a major risk factor for the cancer, and immunocompromised patients, are prone to get anal cancer. In this subgroup, the prognosis is worse, than for non-immunocompromised patients.

Anal cancer is primarily treated with a combination of chemotherapy and radiation. This reduces the need for a colostomy and carries a 5-year survival rate of over 70%. Despite this, the treatment of anal cancer, in this fashion, has not changed since 1974 and surgery is reserved only for patients failing the above therapy. Hence, there exists a need for alternative approaches to treating anal cancer. One such approach is an immunotherapeutic approach using recombinant attenuated live vaccine vectors, such as a Listeria monocytogenes vaccine vector. The present invention provides an attenuated live Listeria vaccine vector for treating anal cancer.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method of treating an anal or vaginal tumor or anal or vaginal cancer in a human subject, the method comprising the step of administering to said subject a combination therapy comprising a chemo-radiation therapy and a recombinant Listeria strain, said Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a heterologous antigen or fragment thereof, thereby treating said anal or vaginal tumor or anal or vaginal cancer in said human subject.

In another aspect, the present invention relates to a method of treating an anal or vaginal neoplasia in a human subject, the method comprising the step of administering to said subject a combination therapy comprising a chemo-radiation therapy and a recombinant Listeria strain, said Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a heterologous antigen or fragment thereof, thereby treating said anal or vaginal neoplasia in said human subject.

In a further aspect, the present invention relates to the use of a composition comprising a recombinant Listeria strain, said Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a heterologous antigen or fragment thereof for treating an anal or vaginal tumor or anal or vaginal cancer in a human subject, the treatment further comprising the step of administering to said subject a chemo-radiation therapy, thereby treating said anal or vaginal tumor or anal or vaginal cancer in said human subject.

In a yet further aspect, the present invention relates to the use of a composition comprising a recombinant Listeria strain, said Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a heterologous antigen or fragment thereof for treating an anal or vaginal neoplasia in a human subject, the treatment further comprising the step of administering to said subject a chemo-radiation therapy, thereby treating said anal or vaginal neoplasia in said human subject.

In a related aspect, the present invention relates to a method of eliciting an anti-tumor cytotoxic T cell response in a human subject comprising administering to said subject said combination therapy.

Other features and advantages disclosed herein will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

FIG. 1. Lm-E7 and Lm-LLO-E7 use different expression systems to express and secrete E7. Lm-E7 was generated by introducing a gene cassette into the orfZ domain of the L. monocytogenes genome (A). The hly promoter drives expression of the hly signal sequence and the first five amino acids (AA) of LLO followed by HPV-16 E7. B), Lm-LLO-E7 was generated by transforming the PrfA-strain XFL-7 with the plasmid pGG-55. pGG-55 has the hly promoter driving expression of a nonhemolytic fusion of LLO-E7. pGG-55 also contains the PrfA gene to select for retention of the plasmid by XFL-7 in vivo.

FIG. 2. Lm-E7 and Lm-LLO-E7 secrete E7. Lm-Gag (lane 1), Lm-E7 (lane 2), Lm-LLO-NP (lane 3), Lm-LLO-E7 (lane 4), XFL-7 (lane 5), and 10403S (lane 6) were grown overnight at 37° C. in Luria-Bertoni broth. Equivalent numbers of bacteria, as determined by OD at 600 nm absorbance, were pelleted and 18 ml of each supernatant was TCA precipitated. E7 expression was analyzed by Western blot. The blot was probed with an anti-E7 mAb, followed by HRP-conjugated anti-mouse (Amersham), then developed using ECL detection reagents.

FIG. 3. Tumor immunotherapeutic efficacy of LLO-E7 fusions. Tumor size in millimeters in mice is shown at 7, 14, 21, 28 and 56 days post tumor-inoculation. Naive mice: open-circles; Lm-LLO-E7: filled circles; Lm-E7: squares; Lm-Gag: open diamonds; and Lm-LLO-NP: filled triangles.

FIG. 4. Splenocytes from Lm-LLO-E7-immunized mice proliferate when exposed to TC-1 cells. C57BL/6 mice were immunized and boosted with Lm-LLO-E7, Lm-E7, or control rLm strains. Splenocytes were harvested 6 days after the boost and plated with irradiated TC-1 cells at the ratios shown. The cells were pulsed with ³H thymidine and harvested. Cpm is defined as (experimental cpm)−(no-TC-1 control).

FIG. 5 A. Induction of E7-specific IFN-gamma-secreting CD8⁺ T cells in the spleens and the numbers penetrating the tumors, in mice administered TC-1 tumor cells and subsequently administered Lm-E7, Lm-LLO-E7, Lm-ActA-E7, or no vaccine (naive). FIG. 5 B. Induction and penetration of E7 specific CD8⁺ cells in the spleens and tumors of the mice described for (A).

FIG. 6. Listeria constructs containing PEST regions induce a higher percentage of E7-specific lymphocytes within the tumor. FIG. 6 A. representative data from 1 experiment. FIG. 6 B. average and SE of data from all 3 experiments.

FIG. 7A. Effect of passaging on bacterial load (virulence) of recombinant Listeria vaccine vectors. Top panel. Lm-Gag. Bottom panel. Lm-LLO-E7. FIG. 7B. Effect of passaging on bacterial load of recombinant Lm-E7 in the spleen. Average CFU of live bacteria per milliliter of spleen homogenate from four mice is depicted.

FIG. 8 shows induction of antigen-specific CD8⁺ T-cells for HIV-Gag and LLO after administration of passaged Lm-Gag versus unpassaged Lm-Gag. Mice were immunized with 10³ (A, B, E, F) or 10⁵ (C, D, G, H) CFU passaged Listeria vaccine vectors, and antigen-specific T-cells were analyzed. B, D, F, H: unpassaged Listeria vaccine vectors. A-D immune response to MHC class I HIV-Gag peptide. E-H: immune response to an LLO peptide. I: splenocytes from mice immunized with 10⁵ CFU passaged Lm-Gag stimulated with a control peptide from HPV E7.

FIG. 9A shows plasmid isolation throughout LB stability study. FIG. 9B shows plasmid isolation throughout TB stability study. FIG. 9C shows quantitation of TB stability study.

FIG. 10 shows numbers of viable bacteria chloramphenicol (CAP)-resistant and CAP-sensitive colony-forming units (CFU) from bacteria grown in LB. Dark bars: CAP⁺; white bars: CAP⁻. The two dark bars and two white bars for each time point represent duplicate samples.

FIG. 11 shows numbers of viable bacteria CAP-resistant and CAP-sensitive CFU from bacteria grown in TB. Dark bars: CAP⁺; white bars: CAP. The two dark bars and two white bars for each time point represent duplicate samples.

FIG. 12. Actual chromatograms showing the region of the D133V mutation (arrows). The mixture ratio is shown in parentheses.

FIG. 13. Representation of the location of the ADV451, 452 and 453 primers and the segment of the PrfA gene amplified in the reaction.

FIG. 14. Specificity of the PCR reaction using primers ADV451 and ADV453.

FIG. 15. Specificity of the PCR reaction using primers ADV452 and ADV453.

FIG. 16. Sensitivity of the PCR reaction to detect the wild-type PrfA sequence using the primer ADV452 and 1 μg as the initial amount of DNA.

FIG. 17. Sensitivity of the PCR reaction to detect the wild-type PrfA sequence using the primer ADV452 and 5 μg as the initial amount of DNA.

FIG. 18. Average density of the bands from the PCR depicted in FIG. 16.

FIG. 19. Average density of the bands from the PCR depicted in FIG. 17.

FIG. 20. Validation of the PCR reaction to detect the wild-type PrfA sequence using the primer ADV452.

FIG. 21. Average density of the bands from the PCR depicted in FIG. 16.

FIG. 22. Analysis of the D133V PrfA mutation in the Lm-LLO-E7. A, Original image used for densitometry; B, Image was digitally enhanced to facilitate the visualization of the low density bands.

FIG. 23. Shows treatment schedule #1 of the patient. The schedule comprises administering a first dose of ADXS-HPV (ADX) before chemo-radiation and the 2-4^(th) doses are given every 28 days after completion of radiation.

FIG. 24. Shows treatment schedule #2 of the patient where the second dose of ADXS-HPV is administered during chemo-radiation.

It will be appreciated that for simplicity and clarity of illustration, elements shown in the Figs. have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the Figs. to indicate corresponding or analogous elements.

DETAILED DESCRIPTION OF THE INVENTION

The present invention disclose, in some embodiments, methods of treating, protecting against, and inducing an immune response against a disease, comprising the step of administering to a subject a recombinant Listeria strain, expressing a fusion peptide comprising a listeriolysin O (LLO) fragment and a heterologous antigen expressed by said disease or fragment thereof. The present invention also provides methods for inducing an anti-disease cytotoxic T-cell (CTL) response in a human subject and treating disorders, and symptoms associated with said disease comprising administration of the recombinant Listeria strain. In one embodiment, provided herein is a recombinant Listeria strain, said recombinant Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising a first an N-terminal fragment of an LLO protein fused to a heterologous antigen or fragment thereof, and wherein said recombinant nucleic acid further comprises a second open reading frame encoding a mutant prfA gene. In one embodiment, the mutant PrfA gene is one that encodes a point mutation from amino acid D or Asp or Aspartate (or Aspartic acid) to amino acid V or Val or Valine at the 133^(rd) amino acid position. In one embodiment, a recombinant Listeria strain disclosed herein comprises a prfA mutation or deletion that is complemented via a plasmid comprised by the same Listeria, wherein the plasmid comprises a mutant prfA gene encoding a mutant PrfA protein comprising a D133V amino acid substitution.

In another embodiment, the recombinant Listeria is an attenuated Listeria. “Attenuation” and “attenuated” may encompass a bacterium, virus, parasite, infectious organism, prion, tumor cell, gene in the infectious organism, and the like, that is modified to reduce toxicity to a host. The host can be a human or animal host, or an organ, tissue, or cell. The bacterium, to give a non-limiting example, can be attenuated to reduce binding to a host cell, to reduce spread from one host cell to another host cell, to reduce extracellular growth, or to reduce intracellular growth in a host cell. Attenuation can be assessed by measuring, e.g., an indicum or indicia of toxicity, the LD₅₀, the rate of clearance from an organ, or the competitive index (see, e.g., Auerbuch, et al. (2001) Infect. Immunity 69:5953-5957). Generally, an attenuation results an increase in the LD₅₀ and/or an increase in the rate of clearance by at least 25%; more generally by at least 50%; most generally by at least 100% (2-fold); normally by at least 5-fold; more normally by at least 10-fold; most normally by at least 50-fold; often by at least 100-fold; more often by at least 500-fold; and most often by at least 1000-fold; usually by at least 5000-fold; more usually by at least 10,000-fold; and most usually by at least 50,000-fold; and most often by at least 100,000-fold.

It will be well appreciated by a skilled artisan that the term “Attenuated gene” may encompass a gene that mediates toxicity, pathology, or virulence, to a host, growth within the host, or survival within the host, where the gene is mutated in a way that mitigates, reduces, or eliminates the toxicity, pathology, or virulence. The reduction or elimination can be assessed by comparing the virulence or toxicity mediated by the mutated gene with that mediated by the non-mutated (or parent) gene. “Mutated gene” encompasses deletions, point mutations, and frameshift mutations in regulatory regions of the gene, coding regions of the gene, non-coding regions of the gene, or any combination thereof. In one embodiment, provided herein is a method of treating an anal or vaginal tumor or anal or vaginal cancer in a human subject, the method comprising the step of administering to said subject a combination therapy comprising a chemo-radiation therapy and a recombinant Listeria strain, said Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a heterologous antigen or fragment thereof, thereby treating said anal or vaginal tumor or anal or vaginal cancer in said human subject.

In one embodiment, said chemo-radiation therapy is administered following a first administration of said recombinant Listeria strain. In another embodiment, said chemo-radiation therapy is administered prior to the administration of said recombinant Listeria strain. In another embodiment, said chemo-radiation therapy is administered following a first administration of said recombinant Listeria strain and prior to one to three booster administrations of said recombinant Listeria strain. In another embodiment, said chemo-radiation therapy is administered concurrently with said recombinant Listeria strain.

In one embodiment, the method disclosed herein comprises administering four doses of a recombinant Listeria provided herein. In one embodiment the recombinant Listeria expresses a fusion protein of N-terminal LLO and a heterologous antigen. In another embodiment, the heterologous antigen is human papilloma virus E7 antigen (HPV-E7). In another embodiment, the HPV antigen is HPVE6.

In one embodiment, the first dose of said recombinant Listeria in the combination therapy provided herein is administered prior to chemo-radiation therapy and the 2^(nd)-4^(th) doses are administered every 28 days after completion of radiation (see Example 11 herein). In another embodiment, the first dose of said recombinant Listeria is administered before chemo-radiation therapy, the second dose of ADXS-HPV is administered during chemo-radiation therapy, and the 3^(rd)-4^(th) doses are administered every 28 days following the completion of chemo-radiation therapy (see Example 11).

In one embodiment, provided herein is a chemo-radiation regiment or chemo-radiation therapy for use in combination with the recombinant Listeria provided herein. In another embodiment, the chemo-radiation therapy provided herein comprises mitomycin and fluorouracil (5-FU) and radiation therapy. In another embodiment, the chemo-radiation therapy can comprise any other chemotherapeutic agents known in the art, including but not limited to, Cyclophosphamide, Mechlorethamine, Chlorambucil, Melphalan, Nitrosoureas, Temozolomide, Azacitidine, Azathioprine, Capecitabine, Cytarabine, Doxifluridine, Gemcitabine, Hydroxyurea, Mercaptopurine, Methotrexate, or Tioguanine (formerly Thioguanine).

In one embodiment, the chemo-radiation regiment or chemo-radiation therapy provided herein comprises administering 2 courses of mitomycin, 5-FU with concurrent radiation (54 Gy in 30 fractions by intensity modulated radiation therapy).

In one embodiment, the radiation provided herein lasts about 6 weeks. In another embodiment, the radiation lasts 3 weeks. In another embodiment, the radiation lasts 4 weeks. In another embodiment, the radiation lasts, 5 weeks. In another embodiment, the radiation lasts 7 weeks. In another embodiment, the radiation lasts 8 weeks. In another embodiment, the radiation lasts 6-8 weeks. In another embodiment, the radiation lasts 4-6 weeks. In another embodiment, the radiation lasts 2-4 weeks. In another embodiment, the radiation lasts 8-10 weeks.

In another embodiment, provided herein is a method of eliciting an anti-tumor cytotoxic T cell response in a human subject comprising administering to said subject said combination therapy.

In one embodiment, provided herein is a method for inducing an immune response against a tumor or a cancer in a human subject, the method comprising the step of administering to said subject a recombinant Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a heterologous antigen or fragment thereof, is, wherein said recombinant nucleic acid further comprises a second open reading frame encoding a mutant prfA gene, thereby inducing an immune response against a tumor or a cancer. In one embodiment, the present invention provides a method of treating a cancer in a human subject, comprising the step of administering to the subject the recombinant Listeria strain provided herein. In another embodiment, the present invention provides a method of protecting a human subject against a cervical cancer, comprising the step of administering to the subject the recombinant Listeria strain provided herein. In another embodiment, the recombinant Listeria strain expresses the recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide. In another embodiment, the method further comprises the step of boosting the human subject with a recombinant Listeria strain of the present invention. In another embodiment, the method further comprises the step of boosting the human subject with an immunogenic composition comprising a heterologous antigen or fragment thereof provided herein. In another embodiment, the method further comprises the step of boosting the human subject with an immunogenic composition that directs a cell of the subject to express the heterologous antigen. In another embodiment, the cell is a tumor cell. In another embodiment, the method further comprises the step of boosting the human subject with the vaccine of the present invention.

In one embodiment, the present invention provides a method of inducing an anti-tumor or an anti-cancer immune response in a human subject, the method comprising the step of administering to said subject a composition comprising a recombinant Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a heterologous antigen or fragment thereof, wherein said recombinant nucleic acid further comprises a second open reading frame encoding a metabolic enzyme, thereby inducing an immune response against a tumor or a cancer. In another embodiment, said Listeria comprises a mutation in the endogenous prfA gene. In another embodiment, the Listeria comprises a mutation or deletion in the endogenous dal/dat and actA genes.

In one embodiment, the nucleic acid molecule provided herein comprises a first open reading frame encoding recombinant polypeptide comprising a heterologous antigen or fragment thereof. In another embodiment, the recombinant polypeptide further comprises a N-terminal LLO fused to the heterologous antigen. In another embodiment, the nucleic acid molecule provided herein further comprises a second open reading frame encoding a metabolic enzyme. In another embodiment, the metabolic enzyme complements an endogenous gene that is lacking in the chromosome of the recombinant Listeria strain. In another embodiment, the metabolic enzyme encoded by the second open reading frame is an alanine racemase enzyme (dal). In another embodiment, the metabolic enzyme encoded by the second open reading frame is a D-amino acid transferase enzyme (dat). In another embodiment, the Listeria strains provided herein comprise a mutation, a deletion or inactivation in the genomic dal, dat, or actA genes. In another embodiment, the Listeria strains provided herein comprise a mutation, a deletion or inactivation in the genomic dal, dat, and actA genes. In another embodiment, the Listeria lack the genomic dal, dat or actA genes. In another embodiment, the Listeria lack the genomic dal, dat and actA genes.

In one embodiment, the fragment thereof in the context of LLO proteins and ActA proteins disclosed herein refer to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues of the LLO or ActA proteins. In another embodiment, the term refers to a peptide or polypeptide comprising an amino acid sequence of at least of at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, at least 250 contiguous amino acid residues of the amino acid sequence, at least 300 contiguous amino acid residues, at least 350 contiguous amino acid residues of, at least 400 contiguous amino acid residues, or at least 450 contiguous amino acid residues of an LLO or ActA protein or polypeptide.

In another embodiment, the fragment is a functional fragment that elicits an immune response against a disease-associated antigen when in the form of an N-terminal LLO/heterologous antigen fusion protein or N-terminal ActA/heterologous antigen fusion protein). In another embodiment, the fragment is an immunogenic fragment. In another embodiment, the fragment is functional in a non-fused form.

The present invention, in certain embodiments, provides codon optimization of a nucleic acid heterologous to Listeria, or of a nucleic acid endogenous to Listeria. The optimal codons utilized by L. monocytogenes for each amino acid are shown US Patent Publication 2007/0207170, which is hereby incorporated by reference herein. A nucleic acid is codon-optimized if at least one codon in the nucleic acid is replaced with a codon that is more frequently used by L. monocytogenes for that amino acid than the codon in the original sequence.

The N-terminal LLO protein fragment and heterologous antigen are, in another embodiment, fused directly to one another. In another embodiment, the genes encoding the N-terminal LLO protein fragment and the heterologous antigen are fused directly to one another. In another embodiment, the N-terminal LLO protein fragment and the heterologous antigen are attached via a linker peptide. In another embodiment, the N-terminal LLO protein fragment and the heterologous antigen are attached via a heterologous peptide. In another embodiment, the N-terminal LLO protein fragment is N-terminal to the heterologous antigen. In another embodiment, the N-terminal LLO protein fragment is the N-terminal-most portion of the fusion protein. As disclosed herein, recombinant Listeria strains expressing LLO-antigen fusions induce anti-tumor immunity (Example 1), elicit antigen-specific T cell proliferation (Example 2), generate antigen-specific, and tumor-infiltrating T cells (Example 3).

In another embodiment, the present invention provides a method of treating a cervical cancer in a human subject, comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising an N-terminal fragment of an LLO protein and an HPV E7 antigen, whereby the recombinant Listeria strain induces an immune response against the E7 antigen, thereby treating a cervical cancer in a human subject. In another embodiment, the recombinant Listeria strain expresses the recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide.

In another embodiment, the present invention provides a method of protecting a human subject against a cervical cancer, comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising an N-terminal fragment of an LLO protein and an HPV E7 antigen, whereby the recombinant Listeria strain induces an immune response against the E7 antigen, thereby protecting a human subject against a cervical cancer. In another embodiment, the recombinant Listeria strain expresses the recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide.

In another embodiment, the present invention provides a method for inducing an immune response against a cervical cancer in a human subject, comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising an N-terminal fragment of an LLO protein and an HPV E7 antigen, thereby inducing an immune response against a cervical cancer in a human subject. In another embodiment, the recombinant Listeria strain expresses the recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide.

In another embodiment, the present invention provides a method of treating a cervical cancer in a human subject, comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising an N-terminal fragment of an ActA protein and heterologous antigen, whereby the recombinant Listeria strain induces an immune response against the heterologous antigen, thereby treating a cervical cancer in a human subject. In another embodiment, the recombinant Listeria strain expresses the recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide.

In another embodiment, the present invention provides a method of protecting a human subject against a cervical cancer, comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising an N-terminal fragment of an ActA protein and a heterologous antigen, whereby the recombinant Listeria strain induces an immune response against the heterologous antigen, thereby protecting a human subject against a cervical cancer. In another embodiment, the recombinant Listeria strain expresses the recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide.

In another embodiment, the present invention provides a method for inducing an immune response against a cervical cancer in a human subject, comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising an N-terminal fragment of an heterologous protein and a heterologous antigen, thereby inducing an immune response against a cervical cancer in a human subject. In another embodiment, the recombinant Listeria strain expresses the recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide.

The N-terminal ActA protein fragment and the heterologous antigen are, in another embodiment, fused directly to one another. In another embodiment, the genes encoding the N-terminal ActA protein fragment and heterologous antigen are fused directly to one another. In another embodiment, the N-terminal ActA protein fragment and heterologous antigen are attached via a linker peptide. In another embodiment, the N-terminal ActA protein fragment and heterologous antigen are attached via a heterologous peptide. In another embodiment, the N-terminal ActA protein fragment is N-terminal to the heterologous antigen. In another embodiment, the N-terminal ActA protein fragment is the N-terminal-most portion of the fusion protein.

In another embodiment, the present invention provides a method of inducing an immune response against a cervical cancer in a human subject, comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising a PEST amino acid sequence-containing peptide and a heterologous antigen, whereby the recombinant Listeria strain induces an immune response against the heterologous antigen, thereby treating a cervical cancer in a human subject. In another embodiment, the recombinant Listeria strain expresses the recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide. In another embodiment, the method protects a human subject against a cervical. In another embodiment, the method treats a cervical cancer in said human subject.

The PEST amino acid amino acid sequence-containing peptide and heterologous antigen are, in another embodiment, fused directly to one another. In another embodiment, the genes encoding the PEST amino acid sequence-containing peptide and heterologous antigen are fused directly to one another. In another embodiment, the PEST amino acid sequence-containing peptide and heterologous antigen are attached via a linker peptide. In another embodiment, the PEST amino acid sequence-containing peptide and heterologous antigen are attached via a heterologous peptide. In another embodiment, the PEST amino acid sequence-containing peptide is N-terminal to the heterologous antigen. In another embodiment, the PEST amino acid sequence-containing peptide is the N-terminal-most portion of the fusion protein.

In another embodiment, the present invention provides a method for vaccinating a human subject against an HPV, comprising the step of administering to the subject the recombinant Listeria strain provided herein, wherein the Listeria expresses an HPV E7 antigen and wherein the Listeria expresses a mutant PrfA gene. In another embodiment, the mutant PrfA gene is a D133V PrfA mutation. In another embodiment, the mutant PrfA gene is in a plasmid in said recombinant Listeria. In another embodiment, the recombinant Listeria strain expresses the recombinant polypeptide. In another embodiment, the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide. In one embodiment, the recombinant Listeria strain comprises a bivalent episomal expression vector, the vector comprising a first, and a second nucleic acid molecule encoding a heterologous antigenic polypeptide or a functional fragment thereof, wherein the first and the second nucleic acid molecules each encode the heterologous antigenic polypeptide or functional fragment thereof in an open reading frame with an N-terminal or truncated or detoxified Listeriolysin O protein (LLO), or a truncated ActA protein, or a PEST amino acid sequence.

In one embodiment, the heterologous antigens expressed by bivalent expression vector are HPV16 E7 and HPV18 E7.

In one embodiment, the recombinant Listeria strain comprises a trivalent episomal expression vector, the vector comprising a first, second, and third nucleic acid molecule, each encoding a heterologous antigenic polypeptide or a functional fragment thereof, wherein the first through third nucleic acid molecules each encode the heterologous antigenic polypeptide or functional fragment thereof in an open reading frame with an N-terminal or truncated or detoxified Listeriolysin O protein (LLO, a truncated ActA protein, or a PEST amino acid sequence.

In one embodiment, the bivalent, trivalent or quadravalent recombinant Listeria strains disclosed herein express at least one heterologous antigen from an open reading frame in a extrachromosomal plasmid or episome. In another embodiment, the bivalent, or trivalent recombinant Listeria strains disclosed herein express at least one heterologous antigen from an open reading frame from at least one extrachromosomal plasmid or episome. In another embodiment, the bivalent recombinant Listeria strains disclosed herein express two heterologous antigens each from an open reading frame of two extrachromosomal plasmids or episomes. In another embodiment, the trivalent recombinant Listeria strains disclosed herein express three heterologous antigens each from an open reading frame of three extrachromosomal plasmids or episomes. In another embodiment, the quadravalent recombinant Listeria strains disclosed herein express four heterologous antigens each from an open reading frame of four extrachromosomal plasmids or episomes.

In another embodiment, the bivalent, trivalent, or quadravalent recombinant Listeria strains disclosed herein express at least one heterologous antigen from an open reading frame in the genome of the Listeria. In another embodiment, the bivalent, trivalent, or quadravalent recombinant Listeria strains provided herein express at least one heterologous antigen from both, an extrachromosomal plasmid or episome, and from the genome of a Listeria provided herein. In another embodiment, each heterologous antigen is expressed in a fusion protein with a PEST-containing polypeptide or peptide provided herein.

In one embodiment, bivalent and multivalent recombinant Listeria encompassed by the present invention include those described in US Pub. No. 2011/0129499, and in US Pub No. 2012/0135033, both of which are incorporated by reference in their entirety herein.

In another embodiment, the subject is at risk for developing an HPV-mediated carcinogenesis (e.g. a cervical cancer). In another embodiment, the subject is HPV-positive. In another embodiment, the subject exhibits cervical intraepithelial neoplasia. In another embodiment, the subject exhibits a squamous intraepithelial lesion. In another embodiment, the subject exhibits a dysplasia in the cervix.

In one embodiment, there heterologous antigen is any tumor associated antigen known in the art and provided herein. In another embodiment, the heterologous antigen is an autoimmune antigen. In another embodiment, the heterologous antigen is an infectious disease antigen. In another embodiment, the heterologous antigen is an HPV-related antigen.

The HPV that is the target of methods disclosed herein is, in another embodiment, an HPV 16. In another embodiment, the HPV is an HPV-18. In another embodiment, the HPV is selected from HPV-16 and HPV-18. In another embodiment, the HPV is an HPV-31. In another embodiment, the HPV is an HPV-35. In another embodiment, the HPV is an HPV-39. In another embodiment, the HPV is an HPV-45. In another embodiment, the HPV is an HPV-51. In another embodiment, the HPV is an HPV-52. In another embodiment, the HPV is an HPV-58. In another embodiment, the HPV is a high-risk HPV type. In another embodiment, the HPV is a mucosal HPV type.

In another embodiment, the present invention provides a method of vaccinating a human subject against an antigen of interest, the method comprising the step of administering intravenously to the human subject a recombinant Listeria strain comprising or expressing the antigen of interest, wherein the first peptide is selected from (a) an N-terminal fragment of an LLO protein; (b) an ActA protein or N-terminal fragment thereof; and (c) a PEST amino acid sequence-containing peptide, thereby vaccinating a human subject against an antigen of interest.

In another embodiment, the present invention provides a method of vaccinating a human subject against an antigen of interest, the method comprising the step of administering intravenously to the human subject an immunogenic composition, comprising a fusion of a first peptide to the antigen of interest, wherein the first peptide is selected from (a) an N-terminal fragment of an LLO protein; (b) an ActA protein or N-terminal fragment thereof; and (c) a PEST amino acid sequence-containing peptide, thereby vaccinating a human subject against an antigen of interest.

In another embodiment, the present invention provides a method of vaccinating a human subject against an antigen of interest, the method comprising the step of administering intravenously to the human subject a recombinant Listeria strain comprising a recombinant polypeptide, the recombinant polypeptide comprising a first peptide fused to the antigen of interest, wherein the first peptide is selected from (a) an N-terminal fragment of an LLO protein; (b) an ActA protein or N-terminal fragment thereof; and (c) a PEST amino acid sequence-containing peptide, thereby vaccinating a human subject against an antigen of interest.

In another embodiment, the present invention provides a method of inducing a CTL response in a human subject against an antigen of interest, the method comprising the step of administering to the human subject a recombinant Listeria strain comprising or expressing the antigen of interest, thereby inducing a CTL response in a human subject against an antigen of interest. In another embodiment, the step of administering is intravenous administration.

As disclosed herein, recombinant Listeria strains expressing LLO-antigen fusions induce anti-tumor immunity (Example 1), elicit antigen-specific T cell proliferation (Example 2), generate antigen-specific, and tumor-infiltrating T cells (Example 3). Thus, vaccines disclosed herein are efficacious at inducing immune responses against E7 and E6.

In another embodiment, the present invention provides a method for inducing a regression of a cancer in a subject, comprising the step of administering to the subject the recombinant Listeria strain provided herein

In another embodiment, the present invention provides a method for reducing an incidence of relapse of a cancer in a subject, comprising the step of administering to the subject the recombinant Listeria strain provided herein.

In another embodiment, the present invention provides a method for suppressing a formation of a tumor in a subject, comprising the step of administering to the subject the recombinant Listeria strain provided herein.

In another embodiment, the present invention provides a method for inducing a remission of a cancer in a subject, comprising the step of administering to the subject the recombinant Listeria strain provided herein.

In another embodiment, the present invention provides a method for impeding a growth of a tumor in a human subject, comprising the step of administering to the subject the recombinant Listeria strain provided herein.

In another embodiment, the present invention provides a method for reducing a size of a tumor in a subject, comprising the step of administering to the subject the recombinant Listeria strain provided herein.

In one embodiment, the disease is an infectious disease, an autoimmune disease, a respiratory disease, a pre-cancerous condition or a cancer.

It will be well appreciated by the skilled artisan that the term “pre-cancerous condition” may encompass dysplasias, preneoplastic nodules; macroregenerative nodules (MRN); low-grade dysplastic nodules (LG-DN); high-grade dysplastic nodules (HG-DN); biliary epithelial dysplasia; foci of altered hepatocytes (FAH); nodules of altered hepatocytes (NAH); chromosomal imbalances; aberrant activation of telomerase; re-expression of the catalytic subunit of telomerase; expression of endothelial cell markers such as CD31, CD34, and BNH9 (see, e.g., Terracciano and Tomillo (2003) Pathologica 95:71-82; Su and Bannasch (2003) Toxicol. Pathol. 31:126-133; Rocken and Carl-McGrath (2001) Dig. Dis. 19:269-278; Kotoula, et al. (2002) Liver 22:57-69; Frachon, et al. (2001) J. Hepatol. 34:850-857; Shimonishi, et al. (2000) J. Hepatobiliary Pancreat. Surg. 7:542-550; Nakanuma, et al. (2003) J. Hepatobiliary Pancreat. Surg. 10:265-281). Methods for diagnosing cancer and dysplasia are disclosed (see, e.g., Riegler (1996) Semin Gastrointest. Dis. 7:74-87; Benvegnu, et al. (1992) Liver 12:80-83; Giannini, et al. (1987) Hepatogastroenterol. 34:95-97; Anthony (1976) Cancer Res. 36:2579-2583).

In one embodiment, an infectious disease is one caused by, but not limited to, any one of the following pathogens: BCG/Tuberculosis, Malaria, Plasmodium falciparum, plasmodium malariae, plasmodium vivax, Rotavirus, Cholera, Diptheria-Tetanus, Pertussis, Haemophilus influenzae, Hepatitis B, Human papilloma virus, Influenza seasonal), Influenza A (H1N1) Pandemic, Measles and Rubella, Mumps, Meningococcus A+C, Oral Polio Vaccines, mono, bi and trivalent, Pneumococcal, Rabies, Tetanus Toxoid, Yellow Fever, Bacillus anthracis (anthrax), Clostridium botulinum toxin (botulism), Yersinia pestis (plague), Variola major (smallpox) and other related pox viruses, Francisella tularensis (tularemia), Viral hemorrhagic fevers, Arenaviruses (LCM, Junin virus, Machupo virus, Guanarito virus, Lassa Fever), Bunyaviruses (Hantaviruses, Rift Valley Fever), Flaviruses (Dengue), Filoviruses (Ebola, Marburg), Burkholderia pseudomallei, Coxiella burnetii (Q fever), Brucella species (brucellosis), Burkholderia mallei (glanders), Chlamydia psittaci (Psittacosis), Ricin toxin (from Ricinus communis), Epsilon toxin of Clostridium perfringens, Staphylococcus enterotoxin B, Typhus fever (Rickettsia prowazekii), other Rickettsias, Food- and Waterborne Pathogens, Bacteria (Diarrheagenic E. coli, Pathogenic Vibrios, Shigella species, Salmonella BCG/, Campylobacter jejuni, Yersinia enterocolitica), Viruses (Caliciviruses, Hepatitis A, West Nile Virus, LaCrosse, Calif. encephalitis, VEE, EEE, WEE, Japanese Encephalitis Virus, Kyasanur Forest Virus, Nipah virus, hantaviruses, Tickborne hemorrhagic fever viruses, Chikungunya virus, Crimean-Congo Hemorrhagic fever virus, Tickborne encephalitis viruses, Hepatitis B virus, Hepatitis C virus, Herpes Simplex virus (HSV), Human immunodeficiency virus (HIV), Human papillomavirus (HPV)), Protozoa (Cryptosporidium parvum, Cyclospora cayatanensis, Giardia lamblia, Entamoeba histolytica, Toxoplasma), Fungi (Microsporidia), Yellow fever, Tuberculosis, including drug-resistant TB, Rabies, Prions, Severe acute respiratory syndrome associated coronavirus (SARS-CoV), Coccidioides posadasii, Coccidioides immitis, Bacterial vaginosis, Chlamydia trachomatis, Cytomegalovirus, Granuloma inguinale, Hemophilus ducreyi, Neisseria gonorrhea, Treponema pallidum, Trichomonas vaginalis, or any other infectious disease known in the art that is not listed herein.

In another embodiment, the infectious disease is a livestock infectious disease. In another embodiment, livestock diseases can be transmitted to man and are called “zoonotic diseases.” In another embodiment, these diseases include, but are not limited to, Foot and mouth disease, West Nile Virus, rabies, canine parvovirus, feline leukemia virus, equine influenza virus, infectious bovine rhinotracheitis (IBR), pseudorabies, classical swine fever (CSF), IBR, caused by bovine herpesvirus type 1 (BHV-1) infection of cattle, and pseudorabies (Aujeszky's disease) in pigs, toxoplasmosis, anthrax, vesicular stomatitis virus, rhodococcus equi, Tularemia, Plague (Yersinia pestis), trichomonas.

In another embodiment, the disease provided herein is a respiratory or inflammatory disease. In another embodiment, the respiratory or inflammatory disease is chronic obstructive pulmonary disease (COPD). In another embodiment, the disease is asthma.

In one embodiment, live attenuated Listeria strains are capable of alleviating asthma symptoms without co-administration of other therapeutic agents, such as anti-inflammatory agents or bronchodilators. In another embodiment, the methods provided herein further comprise the step of co-administering to a subject the live attenuated Listeria strain and one or more therapeutic agents. In another embodiment, the therapeutic agent is an anti-asthmatic agent. In another embodiment, the agent is an anti-inflammatory agent, a non-steroidal anti-inflammatory agent, an antibiotic, an antichlolinerginc agent, a bronchodilator, a corticosteroid, a short-acting beta-agonist, a long-acting beta-agonist, combination inhalers, an antihistamine, or combinations thereof.

In one embodiment, the disease provided herein is a cancer or a tumor. In one embodiment, the tumor is cancerous. In another embodiment, the cancer is breast cancer. In another embodiment, the cancer is a cervical cancer. In another embodiment, the cancer is a Her2 containing cancer. In another embodiment, the cancer is a melanoma. In another embodiment, the cancer is pancreatic cancer. In another embodiment, the cancer is ovarian cancer. In another embodiment, the cancer is gastric cancer. In another embodiment, the cancer is a carcinomatous lesion of the pancreas. In another embodiment, the cancer is pulmonary adenocarcinoma. In another embodiment, it is a glioblastoma multiforme. In another embodiment, the cancer is colorectal adenocarcinoma. In another embodiment, the cancer is pulmonary squamous adenocarcinoma. In another embodiment, the cancer is gastric adenocarcinoma. In another embodiment, the cancer is an ovarian surface epithelial neoplasm (e.g. a benign, proliferative or malignant variety thereof). In another embodiment, the cancer is an oral squamous cell carcinoma. In another embodiment, the cancer is non-small-cell lung carcinoma. In another embodiment, the cancer is an endometrial carcinoma. In another embodiment, the cancer is a bladder cancer. In another embodiment, the cancer is a head and neck cancer. In another embodiment, the cancer is a prostate carcinoma. In another embodiment, the cancer is oropharyngeal cancer. In another embodiment, the cancer is lung cancer. In another embodiment, the cancer is anal cancer. In another embodiment, the cancer is colorectal cancer in another embodiment, the cancer is vaginal cancer. In another embodiment, the cancer is esophageal cancer. The cervical tumor targeted by methods disclosed herein is, in another embodiment, a squamous cell carcinoma. In another embodiment, the cervical tumor is an adenocarcinoma. In another embodiment, the cervical tumor is an adenosquamous carcinoma. In another embodiment, the cervical tumor is a small cell carcinoma. In another embodiment, the cervical tumor is any other type of cervical tumor known in the art.

In one embodiment, the disease provided herein is a neoplasia. In another embodiment, the neoplasia is anal intraepithelial neoplasia (AIN). In another embodiment, the neoplasia is vaginal intraepithelial neoplasia (VIN).

The cervical tumor targeted by methods disclosed herein is, in another embodiment, a squamous cell carcinoma. In another embodiment, the cervical tumor is an adenocarcinoma. In another embodiment, the cervical tumor is an adenosquamous carcinoma. In another embodiment, the cervical tumor is a small cell carcinoma. In another embodiment, the cervical tumor is any other type of cervical tumor known in the art.

In one embodiment, the antigen provided herein is a heterologous tumor antigen, which is also referred to herein as “tumor antigen” “antigenic polypeptide,” or “foreign antigen.” In another embodiment, the antigen is Human Papilloma Virus-E7 (HPV-E7) antigen, which in one embodiment, is from HPV16 (in one embodiment, GenBank Accession No. AAD33253) and in another embodiment, from HPV18 (in one embodiment, GenBank Accession No. P06788). In another embodiment, the antigenic polypeptide is HPV-E6, which in one embodiment, is from HPV16 (in one embodiment, GenBank Accession No. AAD33252, AAM51854, AAM51853, or AAB67615) and in another embodiment, from HPV18 (in one embodiment, GenBank Accession No. P06463). In another embodiment, the antigenic polypeptide is a Her/2-neu antigen. In another embodiment, the antigenic polypeptide is Prostate Specific Antigen (PSA) (in one embodiment, GenBank Accession No. CAD30844, CAD54617, AAA58802, or NP_001639). In another embodiment, the antigenic polypeptide is Stratum Corneum Chymotryptic Enzyme (SCCE) antigen (in one embodiment, GenBank Accession No. AAK69652, AAK69624, AAG33360, AAF01139, or AAC37551). In another embodiment, the antigenic polypeptide is Wilms tumor antigen 1, which in another embodiment is WT-1 Telomerase (GenBank Accession. No. P49952, P22561, NP_659032, CAC39220.2, or EAW68222.1). In another embodiment, the antigenic polypeptide is hTERT or Telomerase (GenBank Accession. No. NM003219 (variant 1), NM198255 (variant 2), NM 198253 (variant 3), or NM 198254 (variant 4). In another embodiment, the antigenic polypeptide is Proteinase 3 (in one embodiment, GenBank Accession No. M29142, M75154, M96839, X55668, NM 00277, M96628 or X56606). In another embodiment, the antigenic polypeptide is Tyrosinase Related Protein 2 (TRP2) (in one embodiment, GenBank Accession No. NP_001913, ABI73976, AAP33051, or Q95119). In another embodiment, the antigenic polypeptide is High Molecular Weight Melanoma Associated Antigen (HMW-MAA) (in one embodiment, GenBank Accession No. NP_001888, AAI28111, or AAQ62842). In another embodiment, the antigenic polypeptide is Testisin (in one embodiment, GenBank Accession No. AAF79020, AAF79019, AAG02255, AAK29360, AAD41588, or NP659206). In another embodiment, the antigenic polypeptide is NY-ESO-1 antigen (in one embodiment, GenBank Accession No. CAA05908, P78358, AAB49693, or NP_640343). In another embodiment, the antigenic polypeptide is PSCA (in one embodiment, GenBank Accession No. AAH65183, NP_005663, NP_082492, 043653, or CAB97347). In another embodiment, the antigenic polypeptide is Interleukin (IL) 13 Receptor alpha (in one embodiment, GenBank Accession No. NP000631, NP001551, NP032382, NP598751, NP001003075, or NP_999506). In another embodiment, the antigenic polypeptide is Carbonic anhydrase IX (CAIX) (in one embodiment, GenBank Accession No. CAI13455, CAI10985, EAW58359, NP_001207, NP_647466, or NP_001101426). In another embodiment, the antigenic polypeptide is carcinoembryonic antigen (CEA) (in one embodiment, GenBank Accession No. AAA66186, CAA79884, CAA66955, AAA51966, AAD15250, or AAA51970). In another embodiment, the antigenic polypeptide is MAGE-A (in one embodiment, GenBank Accession No. NP786885, NP786884, NP005352, NP004979, NP005358, or NP 005353). In another embodiment, the antigenic polypeptide is survivin (in one embodiment, GenBank Accession No. AAC51660, AAY15202, ABF60110, NP001003019, or NP 001082350). In another embodiment, the antigenic polypeptide is GP100 (in one embodiment, GenBank Accession No. AAC60634, YP_655861, or AAB31176). In another embodiment, the antigenic polypeptide is any other antigenic polypeptide known in the art. In another embodiment, the antigenic peptide of the compositions and methods disclosed herein comprise an immunogenic portion of the antigenic polypeptide.

In another embodiment, the antigen is HPV-E6. In another embodiment, the antigen is telomerase (TERT). In another embodiment, the antigen is LMP-1. In another embodiment, the antigen is p53. In another embodiment, the antigen is mesothelin. In another embodiment, the antigen is EGFRVIII. In another embodiment, the antigen is carboxic anhydrase IX (CAIX). In another embodiment, the antigen is PSMA. In another embodiment, the antigen is HMW-MAA. In another embodiment, the antigen is HIV-1 Gag. In another embodiment, the antigen is Tyrosinase related protein 2. In another embodiment, the antigen is selected from HPV-E7, HPV-E6, Her-2, HIV-1 Gag, LMP-1, p53, PSMA, carcinoembryonic antigen (CEA), LMP-1, kallikrein-related peptidase 3 (KLK3), KLK9, Muc, Tyrosinase related protein 2, Muc1, FAP, IL-13R alpha 2, PSA (prostate-specific antigen), gp-100, heat-shock protein 70 (HSP-70), beta-HCG, EGFR-III, Granulocyte colony-stimulating factor (G-CSF), Angiogenin, Angiopoietin-1, Del-1, Fibroblast growth factors: acidic (aFGF) or basic (bFGF), Follistatin, Granulocyte colony-stimulating factor (G-CSF), Hepatocyte growth factor (HGF)/scatter factor (SF), Interleukin-8 (IL-8), Leptin, Midkine, Placental growth factor, Platelet-derived endothelial cell growth factor (PD-ECGF), Platelet-derived growth factor-BB (PDGF-BB), Pleiotrophin (PTN), Progranulin, Proliferin, Transforming growth factor-alpha (TGF-alpha), Transforming growth factor-beta (TGF-beta), Tumor necrosis factor-alpha (TNF-alpha), Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF), VEGFR, VEGFR2 (KDR/FLK-1) or a fragment thereof, FLK-1 or an epitope thereof, FLK-E1, FLK-E2, FLK-I1, endoglin or a fragment thereof, Neuropilin 1 (NRP-1), Angiopoietin 1 (Ang1), Tie2, Platelet-derived growth factor (PDGF), Platelet-derived growth factor receptor (PDGFR), Transforming growth factor-beta (TGF-β), endoglin, TGF-β receptors, monocyte chemotactic protein-1 (MCP-1), VE-cadherin, CD31, ephrin, ICAM-1, V-CAM-1, VAP-1, E-selectin, plasminogen activators, plasminogen activator inhibitor-1, Nitric oxide synthase (NOS), COX-2, AC133, or Id1/Id3, Angiopoietin 3, Angiopoietin 4, Angiopoietin 6, CD105, EDG, HHT1, ORW, ORW1 or a TGFbeta co-receptor, or a combination thereof. In another embodiment, the antigen is a chimeric Her2/neu antigen as disclosed in US Patent Application Publication No. 2011/0142791, which is incorporated by reference herein in its entirety. The use of fragments of antigens provided herein is also encompassed by the present invention.

In another embodiment, the heterologous tumor antigen provided herein is a tumor-associated antigen, which in one embodiment, is one of the following tumor antigens: a MAGE (Melanoma-Associated Antigen E) protein, e.g. MAGE 1, MAGE 2, MAGE 3, MAGE 4, a tyrosinase; a mutant ras protein; a mutant p53 protein; p97 melanoma antigen, a ras peptide or p53 peptide associated with advanced cancers; the HPV 16/18 antigens associated with cervical cancers, KLH antigen associated with breast carcinoma, CEA (carcinoembryonic antigen) associated with colorectal cancer, a MART1 antigen associated with melanoma, or the PSA antigen associated with prostate cancer. In another embodiment, the antigen for the compositions and methods provided herein are melanoma-associated antigens, which in one embodiment are TRP-2, MAGE-1, MAGE-3, gp-100, tyrosinase, HSP-70, beta-HCG, or a combination thereof. It is to be understood that a skilled artisan would be able to use any heterologous antigen not mentioned herein but known in the art for use in the methods and compositions provided herein. It is also to be understood that the present invention provides, but is not limited by, an attenuated Listeria comprising a nucleic acid that encodes at least one of the antigens disclosed herein. The present invention encompasses nucleic acids encoding mutants, muteins, splice variants, fragments, truncated variants, soluble variants, extracellular domains, intracellular domains, mature sequences, and the like, of the disclosed antigens. Provided are nucleic acids encoding epitopes, oligo- and polypeptides of these antigens. Also provided are codon optimized embodiments, that is, optimized for expression in Listeria. The cited references, GenBank Acc. Nos., and the nucleic acids, peptides, and polypeptides disclosed herein, are all incorporated herein by reference in their entirety. In another embodiment, the selected nucleic acid sequence can encode a full length or a truncated gene, a fusion or tagged gene, and can be a cDNA, a genomic DNA, or a DNA fragment, preferably, a cDNA. It can be mutated or otherwise modified as desired. These modifications include codon optimizations to optimize codon usage in the selected host cell or bacteria, i.e. Listeria. The selected sequence can also encode a secreted, cytoplasmic, nuclear, membrane bound or cell surface polypeptide.

In one embodiment, vascular endothelial growth factor (VEGF) is an important signaling protein involved in both vasculogenesis (the formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature). In one embodiment, VEGF activity is restricted mainly to cells of the vascular endothelium, although it does have effects on a limited number of other cell types (e.g. stimulation monocyte/macrophage migration). In vitro, VEGF has been shown to stimulate endothelial cell mitogenesis and cell migration. VEGF also enhances microvascular permeability and is sometimes referred to as vascular permeability factor.

In one embodiment, all of the members of the VEGF family stimulate cellular responses by binding to tyrosine kinase receptors (the VEGFRs) on the cell surface, causing them to dimerize and become activated through transphosphorylation. The VEGF receptors have an extracellular portion consisting of 7 immunoglobulin-like domains, a single transmembrane spanning region and an intracellular portion containing a split tyrosine-kinase domain.

In one embodiment, VEGF-A is a VEGFR-2 (KDR/Flk-1) ligand as well as a VEGFR-1 (Ht-1) ligand. In one embodiment, VEGFR-mediates almost all of the known cellular responses to VEGF. The function of VEGFR-1 is less well defined, although it is thought to modulate VEGFR-2 signaling, in one embodiment, via sequestration of VEGF from VEGFR-2 binding, which in one embodiment, is particularly important during vasculogenesis in the embryo. In one embodiment, VEGF-C and VEGF-D are ligands of the VEGFR-3 receptor, which in one embodiment, mediates lymphangiogenesis.

In one embodiment, the compositions disclosed herein comprise a VEGF receptor or a fragment thereof, which in one embodiment, is a VEGFR-2 and, in another embodiment, a VEGFR-1, and, in another embodiment, VEGFR-3.

In one embodiment, vascular Endothelial Growth Factor Receptor 2 (VEGFR2) is highly expressed on activated endothelial cells (ECs) and participates in the formation of new blood vessels. In one embodiment, VEGFR2 binds all 5 isoforms of VEGF. In one embodiment, signaling of VEGF through VEGFR2 on ECs induces proliferation, migration, and eventual differentiation. In one embodiment, the mouse homologue of VEGFR2 is the fetal liver kinase gene-1 (Elk-1), which is a strong therapeutic target, and has important roles in tumor growth, invasion, and metastasis. In one embodiment, VEGFR2 is also referred to as kinase insert domain receptor (a type III receptor tyrosine kinase) (KDR), cluster of differentiation 309 (CD309), FLK1, Ly73, Krd-1, VEGFR, VEGFR-2, or 6130401C07.

In other embodiments, the antigen is derived from a fungal pathogen, bacteria, parasite, helminth, or viruses. In other embodiments, the antigen is selected from tetanus toxoid, hemagglutinin molecules from influenza virus, diphtheria toxoid, HIV gp120, HIV gag protein, IgA protease, insulin peptide B, Spongospora subterranea antigen, vibriose antigens, Salmonella antigens, pneumococcus antigens, respiratory syncytial virus antigens, Haemophilus influenza outer membrane proteins, Helicobacter pylori urease, Neisseria meningitidis pilins, N. gonorrhoeae pilins, the melanoma-associated antigens (TRP-2, MAGE-1, MAGE-3, gp-100, tyrosinase, MART-1, HSP-70, beta-HCG), human papilloma virus antigens E1 and E2 from type HPV-16, -18, -31, -33, -35 or -45 human papilloma viruses, the tumor antigens CEA, the ras protein, mutated or otherwise, the p53 protein, mutated or otherwise, Muc1, or pSA.

In other embodiments, the antigen is associated with one of the following diseases; cholera, diphtheria, Haemophilus, hepatitis A, hepatitis B, influenza, measles, meningitis, mumps, pertussis, small pox, pneumococcal pneumonia, polio, rabies, rubella, tetanus, tuberculosis, typhoid, Varicella-zoster, whooping cough3 yellow fever, the immunogens and antigens from Addison's disease, allergies, anaphylaxis, Bruton's syndrome, cancer, including solid and blood borne tumors, eczema, Hashimoto's thyroiditis, polymyositis, dermatomyositis, type 1 diabetes mellitus, acquired immune deficiency syndrome, transplant rejection, such as kidney, heart, pancreas, lung, bone, and liver transplants, Graves' disease, polyendocrine autoimmune disease, hepatitis, microscopic polyarteritis, polyarteritis nodosa, pemphigus, primary biliary cirrhosis, pernicious anemia, coeliac disease, antibody-mediated nephritis, glomerulonephritis, rheumatic diseases, systemic lupus erthematosus, rheumatoid arthritis, seronegative spondylarthritides, rhinitis, sjogren's syndrome, systemic sclerosis, sclerosing cholangitis, Wegener's granulomatosis, dermatitis herpetiformis, psoriasis, vitiligo, multiple sclerosis, encephalomyelitis, Guillain-Barre syndrome, myasthenia gravis, Lambert-Eaton syndrome, sclera, episclera, uveitis, chronic mucocutaneous candidiasis, urticaria, transient hypogammaglobulinemia of infancy, myeloma, X-linked hyper IgM syndrome, Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimmune neutropenia, Waldenstrom's macroglobulinemia, amyloidosis, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, malarial circumsporozite protein, microbial antigens, viral antigens, autoantigens, and lesteriosis.

In another embodiment, an HPV E6 antigen is utilized instead of or in addition to an E7 antigen in a method disclosed herein for treating, protecting against, or inducing an immune response against a cervical cancer.

In another embodiment, an ActA protein fragment is utilized instead of or in addition to an LLO fragment in a method disclosed herein for treating, protecting against, or inducing an immune response against a cervical cancer.

In another embodiment, a PEST amino acid sequence-containing protein fragment is utilized instead of or in addition to an LLO fragment in a method disclosed herein for treating, protecting against, or inducing an immune response against a cervical cancer.

In another embodiment, disclosed herein is an immunogenic composition comprising a recombinant Listeria of the present invention. In another embodiment, the immunogenic composition of methods and compositions disclosed herein comprises a recombinant vaccine vector of the present invention. In another embodiment, the immunogenic composition comprises a plasmid of the present invention. In another embodiment, the immunogenic composition comprises an adjuvant. In one embodiment, a vector disclosed herein may be administered as part of a vaccine composition.]

In another embodiment, a vaccine disclosed herein is delivered with an adjuvant. In one embodiment, the adjuvant favors a predominantly Th1-mediated immune response. In another embodiment, the adjuvant favors a Th1-type immune response. In another embodiment, the adjuvant favors a Th1-mediated immune response. In another embodiment, the adjuvant favors a cell-mediated immune response over an antibody-mediated response. In another embodiment, the adjuvant is any other type of adjuvant known in the art. In another embodiment, the immunogenic composition induces the formation of a T cell immune response against a target protein present on a tumor cell.

In another embodiment, the present invention provides a method for inducing an anti-E7 cytotoxic T cell (CTL) response in a human subject, comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising an N-terminal fragment of an LLO protein and an HPV E7 antigen, thereby inducing an anti-E7 CTL response in a human subject. In another embodiment, the recombinant Listeria strain comprises a plasmid that encodes the recombinant polypeptide. In another embodiment, the method further comprises the step of boosting the subject with a recombinant Listeria strain of the present invention. In another embodiment, the method further comprises the step of boosting the subject with an immunogenic composition comprising an E7 antigen. In another embodiment, the method further comprises the step of boosting the subject with an immunogenic composition that directs a cell of the subject to express an E7 antigen. In another embodiment, the CTL response is capable of therapeutic efficacy against an HPV-mediated disease, disorder, or symptom. In another embodiment, the CTL response is capable of prophylactic efficacy against an HPV-mediated disease, disorder, or symptom.

In another embodiment, the present invention provides a method of treating or ameliorating an HPV-mediated disease, disorder, or symptom in a subject, comprising the step of administering to the subject a recombinant Listeria strain, the recombinant Listeria strain comprising a recombinant polypeptide comprising an N-terminal fragment of an LLO protein and an HPV E7 antigen, whereby the recombinant Listeria strain induces an immune response against the E7 antigen, thereby treating or ameliorating an HPV-mediated disease, disorder, or symptom in a subject. In another embodiment, the subject is a human subject. In another embodiment, the subject is a non-human mammal. In another embodiment, the subject is any other type of subject known in the art.

The HPV causing the disease, disorder, or symptom is, in another embodiment, an HPV 16. In another embodiment, the HPV is an HPV-18. In another embodiment, the HPV is an HPV-31. In another embodiment, the HPV is an HPV-35. In another embodiment, the HPV is an HPV-39. In another embodiment, the HPV is an HPV-45. In another embodiment, the HPV is an HPV-51. In another embodiment, the HPV is an HPV-52. In another embodiment, the HPV is an HPV-58. In another embodiment, the HPV is a high-risk HPV type. In another embodiment, the HPV is a mucosal HPV type.

In another embodiment, the HPV-mediated disease, disorder, or symptom is genital warts. In another embodiment, the HPV-mediated disease, disorder, or symptom is non-genital warts. In another embodiment, the HPV-mediated disease, disorder, or symptom is a respiratory papilloma. In another embodiment, the HPV-mediated disease, disorder, or symptom is any other HPV-mediated disease, disorder, or symptom known in the art.

In another embodiment, an HPV E6 antigen is utilized instead of or in addition to an E7 antigen in a method disclosed herein for treating or ameliorating an HPV-mediated disease, disorder, or symptom.

In another embodiment, an ActA protein fragment is utilized instead of or in addition to an LLO fragment in a method disclosed herein for treating or ameliorating an HPV-mediated disease, disorder, or symptom.

In another embodiment, a PEST amino acid sequence-containing protein fragment is utilized instead of or in addition to an LLO fragment in a method disclosed herein for treating or ameliorating an HPV-mediated disease, disorder, or symptom.

In another embodiment, an HPV E6 antigen is utilized instead of or in addition to an E7 antigen in a method disclosed herein for treating or ameliorating an HPV-mediated disease, disorder, or symptom.

The antigen of methods and compositions disclosed herein is, in another embodiment, an HPV E7 protein. In another embodiment, the antigen is an HPV E6 protein. In another embodiment, the antigen is any other HPV protein known in the art.

“E7 antigen” refers, in another embodiment, to an E7 protein. In another embodiment, the term refers to an E7 fragment. In another embodiment, the term refers to an E7 peptide. In another embodiment, the term refers to any other type of E7 antigen known in the art.

The E7 protein of methods and compositions disclosed herein is, in another embodiment, an HPV 16 E7 protein. In another embodiment, the E7 protein is an HPV-18 E7 protein. In another embodiment, the E7 protein is an HPV-31 E7 protein. In another embodiment, the E7 protein is an HPV-35 E7 protein. In another embodiment, the E7 protein is an HPV-39 E7 protein. In another embodiment, the E7 protein is an HPV-45 E7 protein. In another embodiment, the E7 protein is an HPV-51 E7 protein. In another embodiment, the E7 protein is an HPV-52 E7 protein. In another embodiment, the E7 protein is an HPV-58 E7 protein. In another embodiment, the E7 protein is an E7 protein of a high-risk HPV type. In another embodiment, the E7 protein is an E7 protein of a mucosal HPV type.

“E6 antigen” refers, in another embodiment, to an E6 protein. In another embodiment, the term refers to an E6 fragment. In another embodiment, the term refers to an E6 peptide. In another embodiment, the term refers to any other type of E6 antigen known in the art.

The E6 protein of methods and compositions disclosed herein is, in another embodiment, an HPV 16 E6 protein. In another embodiment, the E6 protein is an HPV-18 E6 protein. In another embodiment, the E6 protein is an HPV-31 E6 protein. In another embodiment, the E6 protein is an HPV-35 E6 protein. In another embodiment, the E6 protein is an HPV-39 E6 protein. In another embodiment, the E6 protein is an HPV-45 E6 protein. In another embodiment, the E6 protein is an HPV-51 E6 protein. In another embodiment, the E6 protein is an HPV-52 E6 protein. In another embodiment, the E6 protein is an HPV-58 E6 protein. In another embodiment, the E6 protein is an E6 protein of a high-risk HPV type. In another embodiment, the E6 protein is an E6 protein of a mucosal HPV type.

The immune response induced by methods and compositions disclosed herein is, in another embodiment, a T cell response. In another embodiment, the immune response comprises a T cell response. In another embodiment, the response is a CD8⁺ T cell response. In another embodiment, the response comprises a CD8⁺ T cell response.

In one embodiment, compositions disclosed herein induce a strong innate stimulation of interferon-gamma, which in one embodiment, has anti-angiogenic properties. In one embodiment, a Listeria disclosed herein induces a strong innate stimulation of interferon-gamma, which in one embodiment, has anti-angiogenic properties (Dominiecki et al., Cancer Immunol Immunother. 2005 May; 54(5):477-88. Epub 2004 Oct. 6, incorporated herein by reference in its entirety; Beatty and Paterson, J Immunol. 2001 Feb. 15; 166(4):2276-82, incorporated herein by reference in its entirety). In another embodiment, methods disclosed herein increase a level of interferon-gamma producing cells. In one embodiment, anti-angiogenic properties of Listeria are mediated by CD4⁺ T cells (Beatty and Paterson, 2001). In another embodiment, anti-angiogenic properties of Listeria are mediated by CD8⁺ T cells. In another embodiment, IFN-gamma secretion as a result of Listeria vaccination is mediated by NK cells, NKT cells, Th1 CD4⁺ T cells, TC1 CD8⁺ T cells, or a combination thereof.

In another embodiment, compositions disclosed herein induce production of one or more anti-angiogenic proteins or factors. In one embodiment, the anti-angiogenic protein is IFN-gamma. In another embodiment, the anti-angiogenic protein is pigment epithelium-derived factor (PEDF); angiostatin; endostatin; fms-like tyrosine kinase (sFlt)-1; or soluble endoglin (sEng). In one embodiment, a Listeria disclosed herein is involved in the release of anti-angiogenic factors, and, therefore, in one embodiment, has a therapeutic role in addition to its role as a vector for introducing an antigen to a subject.

In another embodiment, the administration of compositions disclosed herein induces robust systemic antigen-specific immunity. In another embodiment, the administration of compositions disclosed herein induces epitope spreading. In another embodiment, the administration of compositions disclosed herein induces broad-based response to self-derived tumor or neoplasia antigens. In another embodiment the immune response induced by methods and compositions disclosed herein comprises an improvement of the overall balance of suppressor and effector immune cells in the tumor or neoplasia microenvironment (TME). In another embodiment the immune response induced by methods and compositions disclosed herein comprises improvement in the systemic balance of suppressor and effector immunocytes.

In one embodiment, compositions and methods of use thereof disclosed herein generate effector T cells that are able to infiltrate the tumor or neoplasia, destroy tumor cells and eradicate the disease. In another embodiment, methods of use of this invention increase tumor infiltration by T effector cells. In another embodiment, T effector cells comprise CD8+T cells. In another embodiment, T effector cells comprise CD4+T cells.

In one embodiment, tumor infiltrating lymphocytes (TILs) are associated with better prognosis in several tumors, such as colon, ovarian and melanoma. In colon cancer, tumors without signs of micrometastasis have an increased infiltration of immune cells and a Th1 expression profile, which correlate with an improved survival of patients. Moreover, the infiltration of the tumor by T cells has been associated with success of immunotherapeutic approaches in both pre-clinical and human trials. In one embodiment, the infiltration of lymphocytes into the tumor site is dependent on the up-regulation of adhesion molecules in the endothelial cells of the tumor vasculature, generally by proinflammatory cytokines, such as IFN-γ, TNF-α and IL-1. Several adhesion molecules have been implicated in the process of lymphocyte infiltration into tumors, including intercellular adhesion molecule 1 (ICAM-1), vascular endothelial cell adhesion molecule 1 (V-CAM-1), vascular adhesion protein 1 (VAP-1) and E-selectin. However, these cell-adhesion molecules are commonly down-regulated in the tumor vasculature. Thus, in one embodiment, cancer vaccines. As disclosed herein increase TILs, up-regulate adhesion molecules (in one embodiment, ICAM-1, V-CAM-1, VAP-1, E-selectin, or a combination thereof), up-regulate pro-inflammatory cytokines (in one embodiment, IFN-γ, TNF-α, IL-1, or a combination thereof), or a combination thereof.

The N-terminal LLO protein fragment of methods and compositions disclosed herein comprises, in another embodiment, SEQ ID No: 2. In another embodiment, the fragment comprises an LLO signal peptide. In another embodiment, the fragment comprises SEQ ID No: 2. In another embodiment, the fragment consists approximately of SEQ ID No: 2. In another embodiment, the fragment consists essentially of SEQ ID No: 2. In another embodiment, the fragment corresponds to SEQ ID No: 2. In another embodiment, the fragment is homologous to SEQ ID No: 2. In another embodiment, the fragment is homologous to a fragment of SEQ ID No: 2. The ΔLLO used in some of the Examples was 416 AA long (exclusive of the signal sequence), as 88 residues from the amino terminus which is inclusive of the activation domain containing cysteine 484 were truncated. It will be clear to those skilled in the art that any ΔLLO without the activation domain, and in particular without cysteine 484, are suitable for methods and compositions of the present invention. In another embodiment, fusion of an E7 or E6 antigen to any ΔLLO, including the PEST amino acid AA sequence, SEQ ID NO: 1, enhances cell mediated and anti-tumor immunity of the antigen.

The LLO protein utilized to construct vaccines disclosed herein has, in another embodiment, the sequence:

MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIEK KHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSIN QNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQ DNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQL IAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKA VTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKS VSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVP IAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNY DPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWRT VIDDRNLPLVKNRNISIWGTTLYPKYSNKVDNPIE (GenBank Accession No. P13128; SEQ ID NO: 3; nucleic acid sequence is set forth in GenBank Accession No. X15127). The first 25 AA of the proprotein corresponding to this sequence are the signal sequence and are cleaved from LLO when it is secreted by the bacterium. Thus, in this embodiment, the full length active LLO protein is 504 residues long. In another embodiment, the above LLO fragment is used as the source of the LLO fragment incorporated in a vaccine of the present invention.

In another embodiment, the N-terminal fragment of an LLO protein utilized in compositions and methods disclosed herein has the sequence:

(SEQ ID NO: 2) MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPK TPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIV VEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRD SLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYSNV SAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVIS FKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGR QVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGG SAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVI KNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYD.

In another embodiment, the LLO fragment corresponds to about AA 20-442 of an LLO protein utilized herein.

In another embodiment, the LLO fragment has the sequence:

(SEQ ID NO: 4) MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPK TPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIV VEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRD SLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYSNV SAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVIS FKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGR QVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGG SAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVI KNNSEYIETTSKAYTD.

In another embodiment, “truncated LLO” or “ΔLLO” refers to a fragment of LLO that comprises the PEST amino acid domain. In another embodiment, the terms refer to an LLO fragment that comprises a PEST sequence.

In another embodiment, the terms refer to an LLO fragment that does not contain the activation domain at the amino terminus and does not include cysteine 484. In another embodiment, the terms refer to an LLO fragment that is not hemolytic. In another embodiment, the LLO fragment is rendered non-hemolytic by deletion or mutation of the activation domain. In another embodiment, the LLO fragment is rendered non-hemolytic by deletion or mutation of cysteine 484. In another embodiment, the LLO fragment is rendered non-hemolytic by deletion or mutation at another location.

In another embodiment, the LLO fragment consists of about the first 441 AA of the LLO protein. In another embodiment, the LLO fragment consists of about the first 420 AA of LLO. In another embodiment, the LLO fragment is a non-hemolytic form of the LLO protein.

In another embodiment, the LLO fragment contains residues of a homologous LLO protein that correspond to one of the above AA ranges. The residue numbers need not, in another embodiment, correspond exactly with the residue numbers enumerated above; e.g. if the homologous LLO protein has an insertion or deletion, relative to an LLO protein utilized herein, then the residue numbers can be adjusted accordingly.

In another embodiment, the LLO fragment is any other LLO fragment known in the art.

In another embodiment, the recombinant Listeria strain is administered to the human subject at a dose of 1×10⁹-3.31×10¹⁰ CFU. In another embodiment, the dose is 5-500×10⁸ CFU. In another embodiment, the dose is 7-500×10⁸ CFU. In another embodiment, the dose is 10-500×10⁸ CFU. In another embodiment, the dose is 20-500×10⁸ CFU. In another embodiment, the dose is 30-500×10⁸ CFU. In another embodiment, the dose is 50-500×10⁸ CFU. In another embodiment, the dose is 70-500×10⁸ CFU. In another embodiment, the dose is 100-500×10⁸ CFU. In another embodiment, the dose is 150-500×10⁸ CFU. In another embodiment, the dose is 5-300×10⁸ CFU. In another embodiment, the dose is 5-200×10⁸ CFU. In another embodiment, the dose is 5-150×10⁸ CFU. In another embodiment, the dose is 5-100×10⁸ CFU. In another embodiment, the dose is 5-70×10⁸ CFU. In another embodiment, the dose is 5-50×10⁸ CFU. In another embodiment, the dose is 5-30×10⁸ CFU. In another embodiment, the dose is 5-20×10⁸ CFU. In another embodiment, the dose is 1-30×10⁹ CFU. In another embodiment, the dose is 1-20×10⁹ CFU. In another embodiment, the dose is 2-30×10⁹ CFU. In another embodiment, the dose is 1-10×10⁹ CFU. In another embodiment, the dose is 2-10×10⁹ CFU. In another embodiment, the dose is 3-10×10⁹ CFU. In another embodiment, the dose is 2-7×10⁹ CFU. In another embodiment, the dose is 2-5×10⁹ CFU. In another embodiment, the dose is 3-5×10⁹ CFU.

In another embodiment, the dose is 1×10⁹ organisms. In another embodiment, the dose is 1.5×10⁹ organisms. In another embodiment, the dose is 2×10⁹ organisms. In another embodiment, the dose is 3×10⁹ organisms. In another embodiment, the dose is 4×10⁹ organisms. In another embodiment, the dose is 5×10⁹ organisms. In another embodiment, the dose is 6×10⁹ organisms. In another embodiment, the dose is 7×10⁹ organisms. In another embodiment, the dose is 8×10⁹ organisms. In another embodiment, the dose is 10×10⁹ organisms. In another embodiment, the dose is 1.5×10¹⁰ organisms. In another embodiment, the dose is 2×10¹⁰ organisms. In another embodiment, the dose is 2.5×10¹⁰ organisms. In another embodiment, the dose is 3×10¹⁰ organisms. In another embodiment, the dose is 3.3×10¹⁰ organisms. In another embodiment, the dose is 4×10¹⁰ organisms. In another embodiment, the dose is 5×10¹⁰ organisms.

In another embodiment, the recombinant polypeptide of methods disclosed herein is expressed by the recombinant Listeria strain. In another embodiment, the expression is mediated by a nucleotide molecule carried by the recombinant Listeria strain.

In another embodiment, the recombinant Listeria strain expresses the recombinant polypeptide by means of a plasmid that encodes the recombinant polypeptide. In another embodiment, the plasmid comprises a gene encoding a bacterial transcription factor. In another embodiment, the plasmid encodes a Listeria transcription factor. In another embodiment, the transcription factor is PrfA. In another embodiment, the PrfA is a mutant PrfA. In another embodiment, the PrfA mutant protein contains a D133V amino acid mutation.

In another embodiment, the plasmid comprises a gene encoding a metabolic enzyme. In another embodiment, the metabolic enzyme is a bacterial metabolic enzyme. In another embodiment, the metabolic enzyme is a Listerial metabolic enzyme. In another embodiment, the metabolic enzyme is an amino acid metabolism enzyme. In another embodiment, the amino acid metabolism gene is involved in a cell wall synthesis pathway. In another embodiment, the metabolic enzyme is the product of a D-amino acid aminotransferase gene (dat). In another embodiment, the metabolic enzyme is the product of an alanine racemase gene (dal). In another embodiment, the metabolic enzyme is any other metabolic enzyme known in the art.

In another embodiment, a method of present invention further comprises the step of boosting the human subject with a recombinant Listeria strain of the present invention. In another embodiment, the recombinant Listeria strain used in the booster inoculation is the same as the strain used in the initial “priming” inoculation. In another embodiment, the booster strain is different from the priming strain. In another embodiment, the same doses are used in the priming and boosting inoculations. In another embodiment, a larger dose is used in the booster. In another embodiment, a smaller dose is used in the booster.

In another embodiment, a method of present invention further comprises the step of inoculating the human subject with an immunogenic composition comprising the E7 antigen. In another embodiment, the immunogenic composition comprises a recombinant E7 protein or fragment thereof. In another embodiment, the immunogenic composition comprises a nucleotide molecule expressing a recombinant E7 protein or fragment thereof. In another embodiment, the non-Listerial inoculation is administered after the Listerial inoculation. In another embodiment, the non-Listerial inoculation is administered before the Listerial inoculation.

“Boosting” refers, in another embodiment, to administration of an additional vaccine dose to a subject. In another embodiment of methods of the present invention, 2 boosts (or a total of 3 inoculations) are administered. In another embodiment, 3 boosts are administered. In another embodiment, 4 boosts are administered. In another embodiment, 5 boosts are administered. In another embodiment, 6 boosts are administered. In another embodiment, more than 6 boosts are administered.

The recombinant Listeria strain of methods and compositions disclosed herein is, in another embodiment, a recombinant Listeria monocytogenes strain. In another embodiment, the Listeria strain is a recombinant Listeria seeligeri strain. In another embodiment, the Listeria strain is a recombinant Listeria grayi strain. In another embodiment, the Listeria strain is a recombinant Listeria ivanovii strain. In another embodiment, the Listeria strain is a recombinant Listeria murrayi strain. In another embodiment, the Listeria strain is a recombinant Listeria welshimeri strain. In another embodiment, the Listeria strain is a recombinant strain of any other Listeria species known in the art.

The present invention provides a number of listerial species and strains for making or engineering an attenuated Listeria of the present invention. In one embodiment, the Listeria strain is L. monocytogenes 10403S wild type (see Bishop and Hinrichs (1987) J. Immunol. 139: 2005-2009; Lauer, et al. (2002) J. Bact. 184: 4177-4186.) In another embodiment, the Listeria strain is L. monocytogenes DP-L4056 (phage cured) (see Lauer, et al. (2002) J. Bact. 184: 4177-4186). In another embodiment, the Listeria strain is L. monocytogenes DP-L4027, which is phage cured and deleted in the hly gene (see Lauer, et al. (2002) J. Bact. 184: 4177-4186; Jones and Portnoy (1994) Infect. Immunity 65: 5608-5613). In another embodiment, the Listeria strain is L. monocytogenes DP-L4029, which is phage cured, deleted in ActA (see Lauer, et al. (2002) J. Bact. 184: 4177-4186; Skoble, et al. (2000) J. Cell Biol. 150: 527-538). In another embodiment, the Listeria strain is L. monocytogenes DP-L4042 (delta PEST) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. monocytogenes DP-L4097 (LLO-S44A) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. monocytogenes DP-L4364 (delta 1p1A; lipoate protein ligase) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. monocytogenes DP-L4405 (delta in1A) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. monocytogenes DP-L4406 (delta in1B) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. monocytogenes CS-L0001 (delta ActA-delta in1B) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. monocytogenes CS-L0002 (delta ActA-delta 1p1A) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. monocytogenes CS-L0003 (L461T-delta 1p1A) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. monocytogenes DP-L4038 (delta ActA-LLO L461T) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. monocytogenes DP-L4384 (S44A-LLO L461T) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting information). In another embodiment, the Listeria strain is L. monocytogenes. Mutation in lipoate protein (see O'Riordan, et al. (2003) Science 302: 462-464). In another embodiment, the Listeria strain is L. monocytogenes DP-L4017 (10403S hly (L461T), having a point mutation in hemolysin gene (see U.S. Provisional Pat. Appl. Ser. No. 60/490,089 filed Jul. 24, 2003). In another embodiment, the Listeria strain is L. monocytogenes EGD (see GenBank Acc. No. AL591824). In another embodiment, the Listeria strain is L. monocytogenes EGD-e (see GenBank Acc. No. NC_003210. ATCC Acc. No. BAA-679). In another embodiment, the Listeria strain is L. monocytogenes DP-L4029 deleted in uvrAB (see U.S. Provisional Pat. Appl. Ser. No. 60/541,515 filed Feb. 2, 2004; U.S. Provisional Pat. Appl. Ser. No. 60/490,080 filed Jul. 24, 2003). In another embodiment, the Listeria strain is L. monocytogenes ActA-/in1B-double mutant (see ATCC Acc. No. PTA-5562). In another embodiment, the Listeria strain is L. monocytogenes 1plA mutant or hly mutant (see U.S. Pat. Applic. No. 20040013690 of Portnoy, et. al). In another embodiment, the Listeria strain is L. monocytogenes DAL/DAT double mutant. (see U.S. Pat. Applic. No. 20050048081 of Frankel and Portnoy. The present invention encompasses reagents and methods that comprise the above listerial strains, as well as these strains that are modified, e.g., by a plasmid and/or by genomic integration, to contain a nucleic acid encoding one of, or any combination of, the following genes: hly (LLO; listeriolysin); iap (p60); in1A; in1B; in1C; dal (alanine racemase); dat (D-amino acid aminotransferase); plcA; plcB; actA; or any nucleic acid that mediates growth, spread, breakdown of a single walled vesicle, breakdown of a double walled vesicle, binding to a host cell, uptake by a host cell. The present invention is not to be limited by the particular strains disclosed above.

In another embodiment, a recombinant Listeria strain disclosed herein has been passaged through an animal host. In another embodiment, the passaging maximizes efficacy of the strain as a vaccine vector. In another embodiment, the passaging stabilizes the immunogenicity of the Listeria strain. In another embodiment, the passaging stabilizes the virulence of the Listeria strain. In another embodiment, the passaging increases the immunogenicity of the Listeria strain. In another embodiment, the passaging increases the virulence of the Listeria strain. In another embodiment, the passaging removes unstable sub-strains of the Listeria strain. In another embodiment, the passaging reduces the prevalence of unstable sub-strains of the Listeria strain. In another embodiment, the Listeria strain contains a genomic insertion of the gene encoding the antigen-containing recombinant peptide. In another embodiment, the Listeria strain carries a plasmid comprising the gene encoding the antigen-containing recombinant peptide. In another embodiment, the passaging is performed as described herein (e.g. in Example 12). In another embodiment, the passaging is performed by any other method known in the art.

In another embodiment, the recombinant Listeria strain utilized in methods disclosed herein has been stored in a frozen cell bank. In another embodiment, the recombinant Listeria strain has been stored in a lyophilized cell bank.

In another embodiment, the cell bank of methods and compositions disclosed herein is a master cell bank. In another embodiment, the cell bank is a working cell bank. In another embodiment, the cell bank is Good Manufacturing Practice (GMP) cell bank. In another embodiment, the cell bank is intended for production of clinical-grade material. In another embodiment, the cell bank conforms to regulatory practices for human use. In another embodiment, the cell bank is any other type of cell bank known in the art.

“Good Manufacturing Practices” are defined, in another embodiment, by (21 CFR 210-211) of the United States Code of Federal Regulations. In another embodiment, “Good Manufacturing Practices” are defined by other standards for production of clinical-grade material or for human consumption; e.g. standards of a country other than the United States.

In another embodiment, a recombinant Listeria strain utilized in methods disclosed herein is from a batch of vaccine doses.

In another embodiment, a recombinant Listeria strain utilized in methods disclosed herein is from a frozen or lyophilized stock produced by methods provided in U.S. Pat. No. 8,114,414, which is incorporated by reference herein.

In another embodiment, a peptide disclosed herein is a fusion peptide. In another embodiment, “fusion peptide” refers to a peptide or polypeptide comprising 2 or more proteins linked together by peptide bonds or other chemical bonds. In another embodiment, the proteins are linked together directly by a peptide or other chemical bond. In another embodiment, the proteins are linked together with 1 or more AA (e.g. a “spacer”) between the 2 or more proteins.

In another embodiment, a vaccine disclosed herein further comprises an adjuvant. The adjuvant utilized in methods and compositions disclosed herein is, in another embodiment, a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein. In another embodiment, the adjuvant comprises a GM-CSF protein. In another embodiment, the adjuvant is a nucleotide molecule encoding GM-CSF. In another embodiment, the adjuvant comprises a nucleotide molecule encoding GM-CSF. In another embodiment, the adjuvant is saponin QS21. In another embodiment, the adjuvant comprises saponin QS21. In another embodiment, the adjuvant is monophosphoryl lipid A. In another embodiment, the adjuvant comprises monophosphoryl lipid A. In another embodiment, the adjuvant is SBAS2. In another embodiment, the adjuvant comprises SBAS2. In another embodiment, the adjuvant is an unmethylated CpG-containing oligonucleotide. In another embodiment, the adjuvant comprises an unmethylated CpG-containing oligonucleotide. In another embodiment, the adjuvant is an immune-stimulating cytokine. In another embodiment, the adjuvant comprises an immune-stimulating cytokine. In another embodiment, the adjuvant is a nucleotide molecule encoding an immune-stimulating cytokine. In another embodiment, the adjuvant comprises a nucleotide molecule encoding an immune-stimulating cytokine. In another embodiment, the adjuvant is or comprises a quill glycoside. In another embodiment, the adjuvant is or comprises a bacterial mitogen. In another embodiment, the adjuvant is or comprises a bacterial toxin. In another embodiment, the adjuvant is or comprises any other adjuvant known in the art.

In another embodiment, a nucleotide disclosed herein is operably linked to a promoter/regulatory sequence that drives expression of the encoded peptide in the Listeria strain. Promoter/regulatory sequences useful for driving constitutive expression of a gene are well known in the art and include, but are not limited to, for example, the P_(hlyA), P_(ActA), and p60 promoters of Listeria, the Streptococcus bac promoter, the Streptomyces griseus sgiA promoter, and the B. thuringiensis phaZ promoter. In another embodiment, inducible and tissue specific expression of the nucleic acid encoding a peptide disclosed herein is accomplished by placing the nucleic acid encoding the peptide under the control of an inducible or tissue specific promoter/regulatory sequence. Examples of tissue specific or inducible promoter/regulatory sequences which are useful for his purpose include, but are not limited to the MMTV LTR inducible promoter, and the SV40 late enhancer/promoter. In another embodiment, a promoter that is induced in response to inducing agents such as metals, glucocorticoids, and the like, is utilized. Thus, it will be appreciated that the invention includes the use of any promoter/regulatory sequence, which is either known or unknown, and which is capable of driving expression of the desired protein operably linked thereto.

An N-terminal fragment of an ActA protein utilized in methods and compositions disclosed herein has, in another embodiment, the sequence set forth in SEQ ID NO: 5:

MRAMMVVFITANCITINPDHFAATDSEDSSLNTDEWEEEKTEEQPSEVNTGPR YETAREVSSRDIKELEKSNKVRNTNKADLIAMLKEKAEKGPNINNNNSEQTENAA INEEASGADRPAIQVERRHPGLPSDSAAEIKKRRKAIASSDSELESLTYPDKPTKVN KKKVAKESVADASESDLDSSMQSADESSPQPLKANQQPFFPKVFKKIKDAGKWV RDKIDENPEVKKAIVDKSAGLIDQLLTKKKSEEVNASDFPPPPTDEELRLALPETP MLLGFNAPATSEPSSFEFPPPPTDEELRLALPETPMLLGFNAPATSEPSSFEFPPPPT EDELEIIRETASSLDSSFTRGDLASLRNAINRHSQNFSDFPPIPTEEELNGRGGRP. In another embodiment, the ActA fragment comprises the sequence set forth in SEQ ID NO: 5. In another embodiment, the ActA fragment is any other ActA fragment known in the art.

In another embodiment, the recombinant nucleotide encoding a fragment of an ActA protein comprises the sequence set forth in SEQ ID NO: 6:

Atgcgtgcgatgatggtggttttcattactgccaattgcattacgattaaccccgacataatatttgcagcgacagatagcgaa gattctagtctaaacacagatgaatgggaagaagaaaaaacagaagagcaaccaagcgaggtaaatacgggaccaagatacg aaactgcacgtgaagtaagttcacgtgatattaaagaactagaaaaatcgaataaagtgagaaatacgaacaaagcagacctaat agcaatgttgaaagaaaaagcagaaaaaggtccaaatatcaataataacaacagtgaacaaactgagaatgcggctataaatga agaggcttcaggagccgaccgaccagctatacaagtggagcgtcgtcatccaggattgccatcggatagcgcagcggaaatta aaaaaagaaggaaagccatagcatcatcggatagtgagcttgaaagccttacttatccggataaaccaacaaaagtaaataagaa aaaagtggcgaaagagtcagttgcggatgcttctgaaagtgacttagattctagcatgcagtcagcagatgagtcttcaccacaac ctttaaaagcaaaccaacaaccatttttccctaaagtatttaaaaaaataaaagatgcggggaaatgggtacgtgataaaatcgacg aaaatcctgaagtaaagaaagcgattgttgataaaagtgcagggttaattgaccaattattaaccaaaaagaaaagtgaagaggta aatgcttcggacttcccgccaccacctacggatgaagagttaagacttgctttgccagagacaccaatgcttcttggttttaatgctc ctgctacatcagaaccgagctcattcgaatttccaccaccacctacggatgaagagttaagacttgctttgccagagacgccaatg cttcttggttttaatgctcctgctacatcggaaccgagctcgttcgaatttccaccgcctccaacagaagatgaactagaaatcatcc gggaaacagcatcctcgctagattctagttttacaagaggggatttagctagtttgagaaatgctattaatcgccatagtcaaaatttc tctgatttcccaccaatcccaacagaagaagagttgaacgggagaggcggtagacca. In another embodiment, the recombinant nucleotide has the sequence set forth in SEQ ID NO: 6. In another embodiment, the recombinant nucleotide comprises any other sequence that encodes a fragment of an ActA protein.

In another embodiment of the methods and compositions of the present invention, a PEST amino acid AA sequence is fused to the E7 or E6 antigen. As disclosed herein, recombinant Listeria strains expressing PEST amino acid sequence-antigen fusions induce anti-tumor immunity (Example 3) and generate antigen-specific, tumor-infiltrating T cells (Example 4). Further, enhanced cell mediated immunity was demonstrated for fusion proteins comprising an antigen and LLO containing the PEST amino acid AA sequence KENSISSMAPPASPPASPKTPIEKKHADEIDK (SEQ ID NO: 1).

Thus, fusion of an antigen to other LM PEST amino acid sequences and PEST amino acid sequences derived from other prokaryotic organisms will also enhance immunogenicity of the antigen. The PEST amino acid AA sequence has, in another embodiment, a sequence selected from SEQ ID NO: 7-12. In another embodiment, the PEST amino acid sequence is a PEST amino acid sequence from the LM ActA protein. In another embodiment, the PEST amino acid sequence is KTEEQPSEVNTGPR (SEQ ID NO: 7), KASVTDTSEGDLDSSMQSADESTPQPLK (SEQ ID NO: 8), KNEEVNASDFPPPPTDEELR (SEQ ID NO: 9), or RGGIPTSEEFSSLNSGDFTDDENSETTEEEIDR (SEQ ID NO: 10). In another embodiment, the PEST amino acid sequence is from Streptolysin O protein of Streptococcus sp. In another embodiment, the PEST amino acid sequence is from Streptococcus pyogenes Streptolysin O, e.g. KQNTASTETTTTNEQPK (SEQ ID NO: 11) at AA 35-51. In another embodiment, the PEST amino acid sequence is from Streptococcus equisimilis Streptolysin O, e.g. KQNTANTETTTTNEQPK (SEQ ID NO:12) at AA 38-54. In another embodiment, the PEST amino acid sequence is another PEST amino acid AA sequence derived from a prokaryotic organism. In another embodiment, the PEST amino acid sequence is any other PEST amino acid sequence known in the art.

PEST amino acid sequences of other prokaryotic organism can be identified in accordance with methods such as described by, for example Rechsteiner and Rogers (1996, Trends Biochem. Sci. 21:267-271) for LM. Alternatively, PEST amino acid AA sequences from other prokaryotic organisms can also be identified based by this method. Other prokaryotic organisms wherein PEST amino acid AA sequences would be expected to include, but are not limited to, other Listeria species. In another embodiment, the PEST amino acid sequence is embedded within the antigenic protein. Thus, in another embodiment, “fusion” refers to an antigenic protein comprising both the antigen and the PEST amino acid amino acid sequence either linked at one end of the antigen or embedded within the antigen.

In another embodiment, the PEST amino acid sequence is identified using any other method or algorithm known in the art, e.g the CaSPredictor (Garay-Malpartida HM, Occhiucci JM, Alves J, Belizario JE. Bioinformatics. 2005 June; 21 Suppl 1:i169-76). In another embodiment, the following method is used:

A PEST index is calculated for each 30-35 AA stretch by assigning a value of 1 to the amino acids Ser, Thr, Pro, Glu, Asp, Asn, or Gln. The coefficient value (CV) for each of the PEST residue is 1 and for each of the other AA (non-PEST) is 0.

In another embodiment, the LLO protein, ActA protein, or fragment thereof disclosed herein need not be that which is set forth exactly in the sequences set forth herein, but rather other alterations, modifications, or changes can be made that retain the functional characteristics of an LLO or ActA protein fused to an antigen as set forth elsewhere herein. In another embodiment, the present invention utilizes an analog of an LLO protein, ActA protein, or fragment thereof. Analogs differ, in another embodiment, from naturally occurring proteins or peptides by conservative AA sequence differences or by modifications which do not affect sequence, or by both.

In another embodiment, either a whole E7 protein or a fragment thereof is fused to a LLO protein, ActA protein, or PEST amino acid sequence-containing peptide to generate a recombinant peptide of methods of the present invention. The E7 protein that is utilized (either whole or as the source of the fragments) has, in another embodiment, the sequence

MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHY NIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP (SEQ ID No: 13). In another embodiment, the E7 protein is a homologue of SEQ ID No: 13. In another embodiment, the E7 protein is a variant of SEQ ID No: 13. In another embodiment, the E7 protein is an isomer of SEQ ID No: 13. In another embodiment, the E7 protein is a fragment of SEQ ID No: 13. In another embodiment, the E7 protein is a fragment of a homologue of SEQ ID No: 13. In another embodiment, the E7 protein is a fragment of a variant of SEQ ID No: 13. In another embodiment, the E7 protein is a fragment of an isomer of SEQ ID No: 13.

In another embodiment, the sequence of the E7 protein is:

MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARR AEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ (SEQ ID No: 14). In another embodiment, the E6 protein is a homologue of SEQ ID No: 14. In another embodiment, the E6 protein is a variant of SEQ ID No: 14. In another embodiment, the E6 protein is an isomer of SEQ ID No: 14. In another embodiment, the E6 protein is a fragment of SEQ ID No: 14. In another embodiment, the E6 protein is a fragment of a homologue of SEQ ID No: 14. In another embodiment, the E6 protein is a fragment of a variant of SEQ ID No: 14. In another embodiment, the E6 protein is a fragment of an isomer of SEQ ID No: 14.

In another embodiment, the E7 protein has a sequence set forth in one of the following GenBank entries: M24215, NC_004500, V01116, X62843, or M14119. In another embodiment, the E7 protein is a homologue of a sequence from one of the above GenBank entries. In another embodiment, the E7 protein is a variant of a sequence from one of the above GenBank entries. In another embodiment, the E7 protein is an isomer of a sequence from one of the above GenBank entries. In another embodiment, the E7 protein is a fragment of a sequence from one of the above GenBank entries. In another embodiment, the E7 protein is a fragment of a homologue of a sequence from one of the above GenBank entries. In another embodiment, the E7 protein is a fragment of a variant of a sequence from one of the above GenBank entries. In another embodiment, the E7 protein is a fragment of an isomer of a sequence from one of the above GenBank entries.

In another embodiment, either a whole E6 protein or a fragment thereof is fused to a LLO protein, ActA protein, or PEST amino acid sequence-containing peptide to generate a recombinant peptide of methods of the present invention. The E6 protein that is utilized (either whole or as the source of the fragments) has, in another embodiment, the sequence

MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFA FRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCI NCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL (SEQ ID No: 15). In another embodiment, the E6 protein is a homologue of SEQ ID No: 15. In another embodiment, the E6 protein is a variant of SEQ ID No: 15. In another embodiment, the E6 protein is an isomer of SEQ ID No: 15. In another embodiment, the E6 protein is a fragment of SEQ ID No: 15. In another embodiment, the E6 protein is a fragment of a homologue of SEQ ID No: 15. In another embodiment, the E6 protein is a fragment of a variant of SEQ ID No: 15. In another embodiment, the E6 protein is a fragment of an isomer of SEQ ID No: 15.

In another embodiment, the sequence of the E6 protein is:

MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFV VYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPL NPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQV (SEQ ID No: 16). In another embodiment, the E6 protein is a homologue of SEQ ID No: 16. In another embodiment, the E6 protein is a variant of SEQ ID No: 16. In another embodiment, the E6 protein is an isomer of SEQ ID No: 16. In another embodiment, the E6 protein is a fragment of SEQ ID No: 16. In another embodiment, the E6 protein is a fragment of a homologue of SEQ ID No: 16. In another embodiment, the E6 protein is a fragment of a variant of SEQ ID No: 16. In another embodiment, the E6 protein is a fragment of an isomer of SEQ ID No: 16.

In another embodiment, the E6 protein has a sequence set forth in one of the following GenBank entries: M24215, M14119, NC_004500, V01116, X62843, or M14119. In another embodiment, the E6 protein is a homologue of a sequence from one of the above GenBank entries. In another embodiment, the E6 protein is a variant of a sequence from one of the above GenBank entries. In another embodiment, the E6 protein is an isomer of a sequence from one of the above GenBank entries. In another embodiment, the E6 protein is a fragment of a sequence from one of the above GenBank entries. In another embodiment, the E6 protein is a fragment of a homologue of a sequence from one of the above GenBank entries. In another embodiment, the E6 protein is a fragment of a variant of a sequence from one of the above GenBank entries. In another embodiment, the E6 protein is a fragment of an isomer of a sequence from one of the above GenBank entries.

In another embodiment, “homology” refers to identity to an LLO sequence (e.g. to one of SEQ ID No: 2-4) of greater than 70%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 64%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 68%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 72%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 75%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 78%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 80%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 82%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 83%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 85%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 87%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 88%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 90%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 92%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 93%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 95%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 96%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 97%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 98%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of greater than 99%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 2-4 of 100%.

In another embodiment, “homology” refers to identity to an E7 sequence (e.g. to one of SEQ ID No: 13-14) of greater than 70%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 62%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 64%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 68%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 72%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 75%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 78%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 80%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 82%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 83%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 85%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 87%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 88%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 90%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 92%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 93%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 95%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 96%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 97%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 98%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of greater than 99%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 13-14 of 100%.

In another embodiment, “homology” refers to identity to an E6 sequence (e.g. to one of SEQ ID No: 15-16) of greater than 70%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 64%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 68%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 72%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 75%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 78%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 80%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 82%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 83%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 85%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 87%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 88%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 90%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 92%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 93%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 95%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 96%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 97%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 98%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 99%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of 100%.

In another embodiment, “homology” refers to identity to a PEST amino acid sequence (e.g. to one of SEQ ID No: 1, and 7-12) or to an ActA sequence (e.g. to one of SEQ ID No: 5-6) of greater than 70%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 60%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 64%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 68%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 72%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 75%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 78%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 80%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 82%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 83%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 85%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 87%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 88%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 90%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 92%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 93%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 95%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 96%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 97%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 98%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 99%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of 100%.

Protein and/or peptide homology for any AA sequence listed herein is determined, in one embodiment, by methods well described in the art, including immunoblot analysis, or via computer algorithm analysis of AA sequences, utilizing any of a number of software packages available, via established methods. Some of these packages include the FASTA, BLAST, MPsrch or Scanps packages, and employ, in other embodiments, the use of the Smith and Waterman algorithms, and/or global/local or BLOCKS alignments for analysis, for example.

In another embodiment, the LLO protein, ActA protein, or fragment thereof is attached to the antigen by chemical conjugation. In another embodiment, glutaraldehyde is used for the conjugation. In another embodiment, the conjugation is performed using any suitable method known in the art.

In another embodiment, fusion proteins disclosed herein are prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods discussed below. In another embodiment, subsequences are cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments are then ligated, in another embodiment, to produce the desired DNA sequence. In another embodiment, DNA encoding the fusion protein is produced using DNA amplification methods, for example polymerase chain reaction (PCR). First, the segments of the native DNA on either side of the new terminus are amplified separately. The 5′ end of the one amplified sequence encodes the peptide linker, while the 3′ end of the other amplified sequence also encodes the peptide linker. Since the 5′ end of the first fragment is complementary to the 3′ end of the second fragment, the two fragments (after partial purification, e.g. on LMP agarose) can be used as an overlapping template in a third PCR reaction. The amplified sequence will contain codons, the segment on the carboxy side of the opening site (now forming the amino sequence), the linker, and the sequence on the amino side of the opening site (now forming the carboxyl sequence). The insert is then ligated into a plasmid.

In another embodiment, the LLO protein, ActA protein, or fragment thereof and the antigen, or fragment thereof are conjugated by a means known to those of skill in the art. In another embodiment, the antigen, or fragment thereof is conjugated, either directly or through a linker (spacer), to the ActA protein or LLO protein. In another embodiment, the chimeric molecule is recombinantly expressed as a single-chain fusion protein.

In another embodiment, a fusion peptide disclosed herein is synthesized using standard chemical peptide synthesis techniques. In another embodiment, the chimeric molecule is synthesized as a single contiguous polypeptide. In another embodiment, the LLO protein, ActA protein, or fragment thereof; and the antigen, or fragment thereof are synthesized separately, then fused by condensation of the amino terminus of one molecule with the carboxyl terminus of the other molecule, thereby forming a peptide bond. In another embodiment, the ActA protein or LLO protein and antigen are each condensed with one end of a peptide spacer molecule, thereby forming a contiguous fusion protein.

In another embodiment, the peptides and proteins disclosed herein are prepared by solid-phase peptide synthesis (SPPS) as described by Stewart et al. in Solid Phase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford, Ill.; or as described by Bodanszky and Bodanszky (The Practice of Peptide Synthesis, 1984, Springer-Verlag, New York). In another embodiment, a suitably protected AA residue is attached through its carboxyl group to a derivatized, insoluble polymeric support, such as cross-linked polystyrene or polyamide resin. “Suitably protected” refers to the presence of protecting groups on both the alpha-amino group of the amino acid, and on any side chain functional groups. Side chain protecting groups are generally stable to the solvents, reagents and reaction conditions used throughout the synthesis, and are removable under conditions which will not affect the final peptide product. Stepwise synthesis of the oligopeptide is carried out by the removal of the N-protecting group from the initial AA, and couple thereto of the carboxyl end of the next AA in the sequence of the desired peptide. This AA is also suitably protected. The carboxyl of the incoming AA can be activated to react with the N-terminus of the support-bound AA by formation into a reactive group such as formation into a carbodiimide, a symmetric acid anhydride or an “active ester” group such as hydroxybenzotriazole or pentafluorophenly esters.

In another embodiment, the present invention provides a kit comprising vaccine of the present invention, an applicator, and instructional material that describes use of the methods of the invention. Although model kits are described below, the contents of other useful kits will be apparent to the skilled artisan in light of the present disclosure.

EXPERIMENTAL DETAILS SECTION Example 1 LLO-Antigen Fusions Induce Anti-Tumor Immunity Materials and Experimental Methods (Examples 1-2) Cell Lines

The C57BL/6 syngeneic TC-1 tumor was immortalized with HPV-16 E6 and E7 and transformed with the c-Ha-ras oncogene. TC-1, provided by T. C. Wu (Johns Hopkins University School of Medicine, Baltimore, Md.) is a highly tumorigenic lung epithelial cell expressing low levels of with HPV-16 E6 and E7 and transformed with the c-Ha-ras oncogene. TC-1 was grown in RPMI 1640, 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μM nonessential amino acids, 1 mM sodium pyruvate, 50 micromolar (mcM) 2-ME, 400 microgram (mcg)/ml G418, and 10% National Collection Type Culture-109 medium at 37° with 10% CO₂. C3 is a mouse embryo cell from C57BL/6 mice immortalized with the complete genome of HPV 16 and transformed with pEJ-ras. EL-4/E7 is the thymoma EL-4 retrovirally transduced with E7.

L. monocytogenes Strains and Propagation

Listeria strains used were Lm-LLO-E7 (hly-E7 fusion gene in an episomal expression system; FIG. 1A), Lm-E7 (single-copy E7 gene cassette integrated into Listeria genome), Lm-LLO-NP (“DP-L2028”; hly-NP fusion gene in an episomal expression system), and Lm-Gag (“ZY-18”; single-copy HIV-1 Gag gene cassette integrated into the chromosome). E7 was amplified by PCR using the primers 5′-GGCTCGAGCATGGAGATACACC-3′ (SEQ ID No: 17; XhoI site is underlined) and 5′-GGGGACTAGTTTATGGTTTCTGAGAACA-3′ (SEQ ID No: 18; SpeI site is underlined) and ligated into pCR2.1 (Invitrogen, San Diego, Calif.). E7 was excised from pCR2.1 by XhoI/SpeI digestion and ligated into pGG-55. The hly-E7 fusion gene and the pluripotential transcription factor PrfA were cloned into pAM401, a multicopy shuttle plasmid (Wirth R et al, J Bacteriol, 165: 831, 1986), generating pGG-55. The hly promoter drives the expression of the first 441 AA of the hly gene product, (lacking the hemolytic C-terminus, referred to below as “ΔLLO,” and having the sequence set forth in SEQ ID No: 25), which is joined by the XhoI site to the E7 gene, yielding a hly-E7 fusion gene that is transcribed and secreted as LLO-E7. Transformation of a PrfA negative strain of Listeria, XFL-7 (provided by Dr. Hao Shen, University of Pennsylvania), with pGG-55 selected for the retention of the plasmid in vivo (FIGS. 1A-B). The hly promoter and gene fragment were generated using primers 5′-GGGGGCTAGCCCTCCTTTGATTAGTATATTC-3′ (SEQ ID No: 19; NheI site is underlined) and 5′-CTCCCTCGAGATCATAATTTACTTCATC-3′ (SEQ ID No: 20; XhoI site is underlined). The PrfA gene was PCR amplified using primers 5′-GACTACAAGGACGATGACCGACAAGTGATAACCCGGGATCTAAATAAATCCGTT T-3′ (SEQ ID No: 27; XbaI site is underlined) and 5′-CCCGTCGACCAGCTCTTCTTGGTGAAG-3′ (SEQ ID No: 21; SalI site is underlined). Lm-E7 was generated by introducing an expression cassette containing the hly promoter and signal sequence driving the expression and secretion of E7 into the orfZ domain of the LM genome. E7 was amplified by PCR using the primers 5′-GCGGATCCCATGGAGATACACCTAC-3′ (SEQ ID No: 22; BamHI site is underlined) and 5′-GCTCTAGATTATGGTTTCTGAG-3′ (SEQ ID No: 23; XbaI site is underlined). E7 was then ligated into the pZY-21 shuttle vector. LM strain 104035 was transformed with the resulting plasmid, pZY-21-E7, which includes an expression cassette inserted in the middle of a 1.6-kb sequence that corresponds to the orfX, Y, Z domain of the LM genome. The homology domain allows for insertion of the E7 gene cassette into the orfZ domain by homologous recombination. Clones were screened for integration of the E7 gene cassette into the orfZ domain. Bacteria were grown in brain heart infusion medium with (Lm-LLO-E7 and Lm-LLO-NP) or without (Lm-E7 and ZY-18) chloramphenicol (20 μg/ml). Bacteria were frozen in aliquots at −80° C. Expression was verified by Western blotting (FIG. 2).

Western Blotting

Listeria strains were grown in Luria-Bertoni medium at 37° C. and were harvested at the same optical density measured at 600 nm. The supernatants were TCA precipitated and resuspended in 1× sample buffer supplemented with 0.1 N NaOH. Identical amounts of each cell pellet or each TCA-precipitated supernatant were loaded on 4-20% Tris-glycine SDS-PAGE gels (NOVEX, San Diego, Calif.). The gels were transferred to polyvinylidene difluoride and probed with an anti-E7 monoclonal antibody (mAb) (Zymed Laboratories, South San Francisco, Calif.), then incubated with HRP-conjugated anti-mouse secondary Ab (Amersham Pharmacia Biotech, Little Chalfont, U.K.), developed with Amersham ECL detection reagents, and exposed to Hyperfilm (Amersham Pharmacia Biotech).

Measurement of Tumor Growth

Tumors were measured every other day with calipers spanning the shortest and longest surface diameters. The mean of these two measurements was plotted as the mean tumor diameter in millimeters against various time points. Mice were sacrificed when the tumor diameter reached 20 mm Tumor measurements for each time point are shown only for surviving mice.

Effects of Listeria Recombinants on Established Tumor Growth

Six- to 8-wk-old C57BL/6 mice (Charles River) received 2×10⁵ TC-1 cells s.c. on the left flank. One week following tumor inoculation, the tumors had reached a palpable size of 4-5 mm in diameter. Groups of eight mice were then treated with 0.1 LD₅₀ i.p. Lm-LLO-E7 (10⁷ CFU), Lm-E7 (10⁶ CFU), Lm-LLO-NP (10⁷ CFU), or Lm-Gag (5×10⁵ CFU) on days 7 and 14.

⁵¹Cr Release Assay

C57BL/6 mice, 6-8 wk old, were immunized i.p. with 0.1LD₅₀ Lm-LLO-E7, Lm-E7, Lm-LLO-NP, or Lm-Gag. Ten days post-immunization, spleens were harvested. Splenocytes were established in culture with irradiated TC-1 cells (100:1, splenocytes:TC-1) as feeder cells; stimulated in vitro for 5 days, then used in a standard ⁵¹Cr release assay, using the following targets: EL-4, EL-4/E7, or EL-4 pulsed with E7 H-2b peptide (RAHYNIVTF). E:T cell ratios, performed in triplicate, were 80:1, 40:1, 20:1, 10:1, 5:1, and 2.5:1. Following a 4-h incubation at 37° C., cells were pelleted, and 50 μl supernatant was removed from each well. Samples were assayed with a Wallac 1450 scintillation counter (Gaithersburg, Md.). The percent specific lysis was determined as [(experimental counts per minute (cpm)−spontaneous cpm)/(total cpm−spontaneous cpm)]×100.

TC-1-Specific Proliferation

C57BL/6 mice were immunized with 0.1 LD₅₀ and boosted by i.p. injection 20 days later with 1 LD₅₀ Lm-LLO-E7, Lm-E7, Lm-LLO-NP, or Lm-Gag. Six days after boosting, spleens were harvested from immunized and naive mice. Splenocytes were established in culture at 5×10⁵/well in flat-bottom 96-well plates with 2.5×10⁴, 1.25×10⁴, 6×10³, or 3×10³ irradiated TC-1 cells/well as a source of E7 Ag, or without TC-1 cells or with 10 μg/ml Con A. Cells were pulsed 45 h later with 0.5 μCi [³H]thymidine/well. Plates were harvested 18 h later using a Tomtec harvester 96 (Orange, Conn.), and proliferation was assessed with a Wallac 1450 scintillation counter. The change in cpm was calculated as experimental cpm −no Ag cpm.

Flow Cytometric Analysis

C57BL/6 mice were immunized intravenously (i.v.) with 0.1 LD₅₀ Lm-LLO-E7 or Lm-E7 and boosted 30 days later. Three-color flow cytometry for CD8 (53-6.7, PE conjugated), CD62 ligand (CD62L; MEL-14, APC conjugated), and E7 H-2Db tetramer was performed using a FACSCalibur® flow cytometer with CellQuest® software (Becton Dickinson, Mountain View, Calif.). Splenocytes harvested 5 days after the boost were stained at room temperature (rt) with H-2Db tetramers loaded with the E7 peptide (RAHYNIVTF) or a control (HIV-Gag) peptide. Tetramers were used at a 1/200 dilution and were provided by Dr. Larry R. Pease (Mayo Clinic, Rochester, Minn.) and by the NIAID Tetramer Core Facility and the NIH AIDS Research and Reference Reagent Program. Tetramer⁺, CD8⁺, CD62L^(low) cells were analyzed.

B16F0-Ova Experiment

24 C57BL/6 mice were inoculated with 5×10⁵ B16F0-Ova cells. On days 3, 10 and 17, groups of 8 mice were immunized with 0.1 LD₅₀ Lm-OVA (10⁶ cfu), Lm-LLO-OVA (108 cfu) and eight animals were left untreated.

Statistics

For comparisons of tumor diameters, mean and SD of tumor size for each group were determined, and statistical significance was determined by Student's t test. p<0.05 was considered significant.

Results

Lm-E7 and Lm-LLO-E7 were compared for their abilities to impact on TC-1 growth. Subcutaneous tumors were established on the left flank of C57BL/6 mice. Seven days later tumors had reached a palpable size (4-5 mm) Mice were vaccinated on days 7 and 14 with 0.1 LD₅₀ Lm-E7, Lm-LLO-E7, or, as controls, Lm-Gag and Lm-LLO-NP. Lm-LLO-E7 induced complete regression of 75% of established TC-1 tumors, while tumor growth was controlled in the other 2 mice in the group (FIG. 3). By contrast, immunization with Lm-E7 and Lm-Gag did not induce tumor regression. This experiment was repeated multiple times, always with very similar results. In addition, similar results were achieved for Lm-LLO-E7 under different immunization protocols. In another experiment, a single immunization was able to cure mice of established 5 mm TC-1 tumors.

In other experiments, similar results were obtained with 2 other E7-expressing tumor cell lines: C3 and EL-4/E7. To confirm the efficacy of vaccination with Lm-LLO-E7, animals that had eliminated their tumors were re-challenged with TC-1 or EL-4/E7 tumor cells on day 60 or day 40, respectively Animals immunized with Lm-LLO-E7 remained tumor free until termination of the experiment (day 124 in the case of TC-1 and day 54 for EL-4/E7).

Thus, expression of an antigen as a fusion protein with ΔLLO enhances the immunogenicity of the antigen.

Example 2 LM-LLO-E7 Treatment Elicits TC-1 Specific Splenocyte Proliferation

To measure induction of T cells by Lm-E7 with Lm-LLO-E7, TC-1-specific proliferative responses, a measure of antigen-specific immunocompetence, were measured in immunized mice. Splenocytes from Lm-LLO-E7-immunized mice proliferated when exposed to irradiated TC-1 cells as a source of E7, at splenocyte: TC-1 ratios of 20:1, 40:1, 80:1, and 160:1 (FIG. 4). Conversely, splenocytes from Lm-E7 and rLm control-immunized mice exhibited only background levels of proliferation.

Example 3 Fusion of E7 to LLO, actA, or a Pest Amino Acid Sequence Enhances E7-Specific Immunity and Generates Tumor-Infiltrating E7-Specific CD8⁺ Cells Materials and Experimental Methods

500 mcl (microliter) of MATRIGEL®, comprising 100 mcl of 2×10⁵ TC-1 tumor cells in phosphate buffered saline (PBS) plus 400 mcl of MATRIGEL® (BD Biosciences, Franklin Lakes, N.J.) were implanted subcutaneously on the left flank of 12 C57BL/6 mice (n=3). Mice were immunized intraperitoneally on day 7, 14 and 21, and spleens and tumors were harvested on day 28. Tumor MATRIGELs were removed from the mice and incubated at 4° C. overnight in tubes containing 2 milliliters (ml) of RP 10 medium on ice. Tumors were minced with forceps, cut into 2 mm blocks, and incubated at 37° C. for 1 hour with 3 ml of enzyme mixture (0.2 mg/ml collagenase-P, 1 mg/ml DNAse-1 in PBS). The tissue suspension was filtered through nylon mesh and washed with 5% fetal bovine serum+0.05% of NaN₃ in PBS for tetramer and IFN-gamma staining.

Splenocytes and tumor cells were incubated with 1 micromole (mcm) E7 peptide for 5 hours in the presence of brefeldin A at 10⁷ cells/ml. Cells were washed twice and incubated in 50 mcl of anti-mouse Fc receptor supernatant (2.4 G2) for 1 hour or overnight at 4° C. Cells were stained for surface molecules CD8 and CD62L, permeabilized, fixed using the permeabilization kit Golgi-Stop® or Golgi-Plug® (Pharmingen, San Diego, Calif.), and stained for IFN-gamma. 500,000 events were acquired using two-laser flow cytometer FACSCalibur and analyzed using Cellquest Software (Becton Dickinson, Franklin Lakes, N.J.). Percentages of IFN-gamma secreting cells within the activated (CD62L^(low)) CD8⁺ T cells were calculated.

For tetramer staining, H-2D^(b) tetramer was loaded with phycoerythrin (PE)-conjugated E7 peptide (RAHYNIVTF, SEQ ID NO: 24), stained at rt for 1 hour, and stained with anti-allophycocyanin (APC) conjugated MEL-14 (CD62L) and FITC-conjugated CD8 at 4° C. for 30 min Cells were analyzed comparing tetramer⁺ CD8⁺ CD62L^(low) cells in the spleen and in the tumor.

Results

To analyze the ability of Lm-ActA-E7 to enhance antigen specific immunity, mice were implanted with TC-1 tumor cells and immunized with either Lm-LLO-E7 (1×10⁷ CFU), Lm-E7 (1×10⁶ CFU), or Lm-ActA-E7 (2×10⁸ CFU), or were untreated (naïve). Tumors of mice from the Lm-LLO-E7 and Lm-ActA-E7 groups contained a higher percentage of IFN-gamma-secreting CD8⁺ T cells (FIG. 5A) and tetramer-specific CD8⁺ cells (FIG. 5B) than in Lm-E7 or naive mice.

In another experiment, tumor-bearing mice were administered Lm-LLO-E7, Lm-PEST-E7, Lm-ΔPEST-E7, or Lm-E7epi, and levels of E7-specific lymphocytes within the tumor were measured. Mice were treated on days 7 and 14 with 0.1 LD₅₀ of the 4 vaccines. Tumors were harvested on day 21 and stained with antibodies to CD62L, CD8, and with the E7/Db tetramer. An increased percentage of tetramer-positive lymphocytes within the tumor were seen in mice vaccinated with Lm-LLO-E7 and Lm-PEST-E7 (FIG. 6A). This result was reproducible over three experiments (FIG. 6B).

Thus, Lm-LLO-E7, Lm-ActA-E7, and Lm-PEST-E7 are each efficacious at induction of tumor-infiltrating CD8⁺ T cells and tumor regression.

Example 4 Passaging of Listeria Vaccine Vectors Through Mice Elicits Increased Immune Responses to Heterologous and Endogenous Antigens Materials and Experimental Methods Bacterial Strains

L. monocytogenes strain 10403S, serotype 1 (ATCC, Manassas, Va.) was the wild type organism used in these studies and the parental strain of the constructs described below. Strain 10403S has an LD₅₀ of approximately 5×10⁴ CFU when injected intraperitoneally into BALB/c mice. “Lm-Gag” is a recombinant LM strain containing a copy of the HIV-1 strain HXB (subtype B laboratory strain with a syncytia-forming phenotype) gag gene stably integrated into the listerial chromosome using a modified shuttle vector pKSV7. Gag protein was expressed and secreted by the strain, as determined by Western blot. All strains were grown in brain-heart infusion (BHI) broth or agar plates (Difco Labs, Detroit, Mich.).

Bacterial Culture

Bacteria from a single clone expressing the passenger antigen and/or fusion protein were selected and cultured in BHI broth overnight. Aliquots of this culture were frozen at −70° C. with no additives. From this stock, cultures were grown to 0.1-0.2 O.D. at 600 nm, and aliquots were again frozen at −70° C. with no additives. To prepare cloned bacterial pools, the above procedure was used, but after each passage a number of bacterial clones were selected and checked for expression of the target antigen, as described herein. Clones in which expression of the foreign antigen was confirmed were used for the next passage.

Passage of Bacteria in Mice

6-8 week old female BALB/c (H-2d) mice were purchased from Jackson Laboratories (Bar Harbor, Me.) and were maintained in a pathogen-free microisolator environment. The titer of viable bacteria in an aliquot of stock culture, stored frozen at −70° C., was determined by plating on BHI agar plates on thawing and prior to use. In all, 5×10⁵ bacteria were injected intravenously into BALB/c mice. After 3 days, spleens were harvested, homogenized, and serial dilutions of the spleen homogenate were incubated in BHI broth overnight and plated on BHI agar plates. For further passage, aliquots were again grown to 0.1-0.2 O.D., frozen at −70° C., and bacterial titer was again determined by serial dilution. After the initial passage (passage 0), this sequence was repeated for a total of 4 times.

Intracellular Cytokine Stain for IFN-Gamma

Lymphocytes were cultured for 5 hours in complete RPMI-10 medium supplemented with 50 U/ml human recombinant IL-2 and 1 microliter/ml Brefeldin A (Golgistop™; PharMingen, San Diego, Calif.) in the presence or absence of either the cytotoxic T-cell (CTL) epitope for HIV-GAG (AMQMLKETI; SEQ ID No: 25), Listeria LLO (GYKDGNEYI; SEQ ID No: 26) or the HPV virus gene E7 (RAHYNIVTF (SEQ ID No: 24), at a concentration of 1 micromole. Cells were first surface-stained, then washed and subjected to intracellular cytokine stain using the Cytofix/Cytoperm kit in accordance with the manufacturer's recommendations (PharMingen, San Diego, Calif.). For intracellular IFN-gamma stain, FITC-conjugated rat anti-mouse IFN-gamma monoclonal antibody (clone XMG 1.2) and its isotype control Ab (rat IgG1; both from PharMingen) was used. In all, 10⁶ cells were stained in PBS containing 1% Bovine Serum Albumin and 0.02% sodium azide (FACS Buffer) for 30 minutes at 4° C. followed by 3 washes in FACS buffer. Sample data were acquired on either a FACScan™ flowcytometer or FACSCalibur™ instrument (Becton Dickinson, San Jose, Calif.). Three-color flow cytometry for CD8 (PERCP conjugated, rat anti-mouse, clone 53-6.7 Pharmingen, San Diego, Calif.), CD62L (APC conjugated, rat anti-mouse, clone MEL-14), and intracellular IFN-gamma was performed using a FACSCalibur™ flow cytometer, and data were further analyzed with CELLQuest software (Becton Dickinson, Mountain View, Calif.). Cells were gated on CD8 high and CD62L^(low) before they were analyzed for CD8⁺ and intracellular IFN-gamma staining.

Results Passaging in Mice Increases the Virulence of Recombinant Listeria Monocytogenes

Three different constructs were used to determine the impact of passaging on recombinant Listeria vaccine vectors. Two of these constructs carry a genomic insertion of the passenger antigen: the first comprises the HIV gag gene (Lm-Gag), and the second comprises the HPV E7 gene (Lm-E7). The third (Lm-LLO-E7) comprises a plasmid with the fusion gene for the passenger antigen (HPV E7) fused with a truncated version of LLO and a gene encoding PrfA, the positive regulatory factor that controls Listeria virulence factors. This plasmid was used to complement a PrfA negative mutant so that in a live host, selection pressures would favor conservation of the plasmid, because without it the bacterium is avirulent. All 3 constructs had been propagated extensively in vitro for many bacterial generations.

Passaging the bacteria resulted in an increase in bacterial virulence, as measured by numbers of surviving bacteria in the spleen, with each of the first 2 passages. For Lm-Gag and Lm-LLO-E7, virulence increased with each passage up to passage 2 (FIG. 7A). The plasmid-containing construct, Lm-LLO-E7, demonstrated the most dramatic increase in virulence. Prior to passage, the initial immunizing dose of Lm-LLO-E7 had to be increased to 10⁷ bacteria and the spleen had to be harvested on day 2 in order to recover bacteria (whereas an initial dose of 10⁵ bacteria for Lm-Gag was harvested on day 3). After the initial passage, the standard dosage of Lm-LLO-E7 was sufficient to allow harvesting on day 3. For Lm-E7, virulence increased by 1.5 orders of magnitude over unpassaged bacteria (FIG. 7B).

Thus, passage through mice increases the virulence of Listeria vaccine strains.

Passaging Increases the Ability of L. monocytogenes to Induce CD8⁺ T Cells

Next, the effect of passaging on induction of antigen-specific CD8⁺ T cells was determined by intracellular cytokine staining with immunodominant peptides specific for MHC-class I using HIV-Gag peptide AMQMLKETI (SEQ ID No: 25) and LLO 91-99 (GYKDGNEYI; SEQ ID No: 26). Injection of 10³ CFU passaged bacteria (Lm-Gag) into mice elicited significant numbers of HIV-Gag-specific CD8⁺ T cells, while the same dose of non-passaged Lm-Gag induced no detectable Gag-specific CD8⁺ T cells. Even increasing the dose of unpassaged bacteria 100-fold did not compensate for their relative avirulence; in fact, no detectable Gag-specific CD8⁺ T cells were elicited even at the higher dose. The same dose increase with passaged bacteria increased Gag-specific T cell induction by 50% (FIG. 8). The same pattern of induction of antigen-specific CD8⁺ T cells was observed with LLO-specific CD8⁺ T cells, showing that these results were not caused by the properties of the passenger antigen, since they were observed with LLO, an endogenous Listeria antigen.

Thus, passage through mice increases the immunogenicity of Listeria vaccine strains.

Example 5 A prfA-Containing Plasmid is Stable in an LM Strain with a prfA Deletion in the Absence of Antibiotics Materials and Experimental Methods Bacteria

L. monocytogenes strain XFL7 contains a 300 base pair deletion in the PrfA gene XFL7 carries pGG55 which partially restores virulence and confers CAP resistance, and is described in United States Patent Application Publication No. 200500118184.

Development of Protocol for Plasmid Extraction from Listeria

1 mL of Listeria monocytogenes Lm-LLO-E7 research working cell bank vial was inoculated into 27 mL BH1 medium containing 34 μg/mL CAP and grown for 24 hours at 37° C. and 200 rpm.

Seven 2.5 mL samples of the culture were pelleted (15000 rpm for 5 minutes), and pellets were incubated at 37° C. with 50 μl lysozyme solution for varying amounts of time, from 0-60 minutes.

Lysozyme solution:

-   -   29 μl 1 M dibasic Potassium Phosphate     -   21 μl 1 M monobasic Potassium Phosphate     -   500 μl 40% Sucrose (filter sterilized through 0.45/μm filter)     -   450 μl water     -   60 μl lysozyme (50 mg/mL)

After incubation with the lysozyme, the suspensions were centrifuged as before and the supernatants discarded. Each pellet was then subjected to plasmid extraction by a modified version of the QIAprep Spin Miniprep Kit® (Qiagen, Germantown, Md.) protocol. The changes to the protocol were as follows:

-   -   1. The volumes of buffers PI, P2 and N3 were all increased         threefold to allow complete lysis of the increased biomass.     -   2. 2 mg/mL of lysozyme was added to the resuspended cells before         the addition of P2. The lysis solution was then incubated at         37° C. for 15 minutes before neutralization.     -   3. The plasmid DNA was resuspended in 30 μL rather than 50 μL to         increase the concentration.

In other experiments, the cells were incubated for 15 min in P1 buffer+Lysozyme, then incubated with P2 (lysis buffer) and P3 (neutraliztion buffer) at room temperature.

Equal volumes of the isolated plasmid DNA from each subculture were run on an 0.8% agarose gel stained with ethidium bromide and visualized for any signs of structural or segregation instability.

The results showed that plasmid extraction from L. monocytogenes Lm-LLO-E7 increases in efficiency with increasing incubation time with lysozyme, up to an optimum level at approximately 50 minutes incubation. 11002251 These results provide an effective method for plasmid extraction from Listeria vaccine strains.

Replica Plating

Dilutions of the original culture were plated onto plates containing LB or TB agar in the absence or presence of 34 μg/mL CAP. The differences between the counts on selective and non-selective agar were used to determine whether there was any gross segregational instability of the plasmid.

Results

The genetic stability (i.e. the extent to which the plasmid is retained by or remains stably associated with the bacteria in the absence of selection pressure; e.g. antibiotic selection pressure) of the pGG55 plasmid in L. monocytogenes strain XFL7 in the absence of antibiotic was assessed by serial sub-culture in both Luria-Bertani media (LB: 5 g/L NaCl, 10 g/ml soy peptone, 5 g/L yeast extract) and Terrific Broth media (TB: 10 g/L glucose, 11.8 g/L soy peptone, 23.6 g/L yeast extract, 2.2 g/L KH₂PO₄, 9.4 g/L K₂HPO₄), in duplicate cultures. 50 mL of fresh media in a 250 mL baffled shake flask was inoculated with a fixed number of cells (1 ODmL), which was then subcultured at 24 hour intervals. Cultures were incubated in an orbital shaker at 37° C. and 200 rpm. At each subculture the OD₆₀₀ was measured and used to calculate the cell doubling time (or generation) elapsed, until 30 generations were reached in LB and 42 in TB. A known number of cells (15 ODmL) at each subculture stage (approximately every 4 generations) were pelleted by centrifugation, and the plasmid DNA was extracted using the Qiagen QIAprep Spin Miniprep® protocol described above. After purification, plasmid DNA was subjected to agarose gel electrophoresis, followed by ethidium bromide staining. While the amount of plasmid in the preps varied slightly between samples, the overall trend was a constant amount of plasmid with respect to the generational number of the bacteria (FIGS. 9A-B). Thus, pGG55 exhibited stability in strain XFL7, even in the absence of antibiotic.

Plasmid stability was also monitored during the stability study by replica plating on agar plates at each stage of the subculture. Consistent with the results from the agarose gel electrophoresis, there was no overall change in the number of plasmid-containing cells throughout the study in either LB or TB liquid culture (FIGS. 10 and 11, respectively).

These findings demonstrate that PrfA-encoding plasmids exhibit stability in the absence of antibiotic in Listeria strains containing mutations in rfA gene.

Materials and Methods (Examples 6-10) PCR Reagents

The primers used for amplification of the PrfA gene and discrimination of the D133V mutation are shown in Table 1. Stock solutions of the primers ADV451, 452 and 453 were prepared by diluting the primers in TE buffer to 400 μM. An aliquot of the stock solution was further diluted to 20 μM in water (PCR grade) to prepare a working solution. Primers were stored at −20° C. The reagents used in the PCR are shown in Table 2.

TABLE 1  Primers ADV451, 452 and 453. Primer Orientation Sequence (5′→3′) Specificity ADV451 Forward CCTAGCTAAATTTAATGT D133V (SEQ ID NO: 28) mutation ADV452 Forward CCTAGCTAAATTTAATGA Wild-type (SEQ ID NO: 29) sequence ADV453 Reverse TAATTTTCCCCAAGTAGC Shared AGG sequence (SEQ ID NO: 30)

TABLE 2 PCR reagents. Catalog Description Provider number 1 0.2 ml thin-walled PCR tubes: Applied N801-0612 GeneAmp autoclaved reaction Biosystems tube with cap 2 Water (PCR reagent) Sigma W1754 3 Taq DNA Polymerase with 10x reaction Sigma D1806 buffer containing 15 mM MgCl₂ 4 Set of deoxynucleotides (dNTPs), Sigma D7295 10 mM each 5 Primers ADV451, ADV452 Invitrogen and ADV453 6 Template DNA, midipreparations of pGG55 plasmids 7 Thermal cycler PTC200 MJ Research (48 wells block)

Plasmid DNA Preparation

pGG55 plasmids with (pGG55 D133V) and without (pGG55 WT) the prfA mutation were extracted and purified by midipreparations either from E. coli or Listeria monocytogenes using the PureLink™ HiPure Plasmid Midiprep Kit (Invitrogen, K2100-05), according to the manufacturer's instructions. For plasmid purification from Listeria, bacterial strains carrying the pGG55 D133V or WT plasmids were streak plated from frozen stocks in BHI agar plates supplemented with chloramphenicol (25 μg/ml). A single colony from each strain was grown in 5 ml of selective medium (BHI broth with 25 μg/ml of chloramphenicol) for 6 hours with vigorous shaking at 37° C. and subinoculated 1:500 in 100 ml of selective medium for overnight growth under similar conditions. Bacteria from the overnight culture were harvested by centrifugation at 4,000×g for 10 minutes and resuspended buffer R3 (resuspension buffer) containing 2 mg/ml of lysozyme (Sigma, L7001). The bacteria suspension was incubated for at least 1 hour at 37° C. before proceeding to the regular protocol. Concentration and purity of the eluted plasmids were measured in a spectrophotometer at 260 nm and 280 nm. To prepare the template DNAs, the pGG55 D133V and WT plasmids were resuspended in water to a final concentration of 1 μg/μl from the midiprep stock solution. For the pGG55 WT plasmid, serial 10-fold dilutions from the 1 μg/μl solution were prepared, corresponding to dilutions from 10⁻¹ to 10⁻⁷.

prfA Specific PCR Protocol to Test Clinical Grade Material

The reaction mixture contained 1×PCR buffer, 1.5 mM MgCl₂, 0.8 mM dNTPs, 0.4 μM of each primer, 0.05 U/μl of Taq DNA polymerase and 0.04 μg/μl of the pGG55 D133V template plasmid. For each test, 10 tubes were required and the key components in each tube in a 25 μl reaction are shown in the Table 3. For the PCR reaction, a master mix was prepared with enough reagents for 11 reactions as shown in Table 4, and 24 μl of this PCR mix was added to each tube. Subsequently, a total of 1 μl of the serially diluted pGG55 WT plasmid was added to the corresponding tubes: 1 μg in tube 3; 100 pg in tube 4; 10 pg in tube 5; 1 pg in tube 6; 100 fg in tube 7; 10 fg in tube 8; 1 fg in tube 9; 0.1 fg in tube 10. This serial dilution was used to calibrate a standard curve to determine the method sensitivity. Additionally, 0.5 μl of water and 0.5 μl of primer ADV451 (20 μM stock) were added in tube 1, and 1 μl of water added in tube 2, completing 25 μl of final volume. The quantities of each reagent per tube for a 25 μl reaction are shown in Table 5. The PCR cycling conditions used in the reaction are shown in Table 6.

After conclusion of the PCR reaction, 5 μl of gel-loading buffer (6×, with bromophenol blue) was added to each sample and 10 μl were analyzed by electrophoresis in 1.2% agarose gel in TBE buffer. The gel dimensions were 7 cm×7 cm×1 cm with a 15 sample wells (1 mm×2 mm) comb. The gel was run at 100 V for ˜30 minutes, until the bromophenol blue dye reached the middle of the gel. The gel was stained in ethidium bromide (0.5 μg/ml) for 20 minutes, destaining in water for 10 minutes. The gel is visualized by illumination with UV light and photographed. The image was analyzed using a band densitometry software (Quantity One version 4.5.1, BioRad).

TABLE 3 Set of individual PCR reactions to validate the method to detect the presence of wild-type prfA sequence in Lm-LLO-E7 samples. Expected Tube Primer A Primer B Template DNA Function result 1 ADV451 ADV453 1 ng of pGG55 Positive control for Positive (D133V) the ADV451 reaction 2 ADV452 ADV453 1 ng of pGG55 Negative control for Negative (D133V) the ADV452 reaction (specificity) 3 ADV452 ADV453 1 ng of pGG55 Positive control for Positive (wild-type) + 1 ng the ADV452 reaction of pGG55 (D133V) 4 ADV452 ADV453 100 pg of pGG55 Test the sensitivity of Positive (wild-type) + 1 ng the reaction of pGG55 (D133V) 5 ADV452 ADV453 10 pg of pGG55 Test the sensitivity of Positive (wild-type) + 1 ng the reaction of pGG55 (D133V) 6 ADV452 ADV453 1 pg of pGG55 Test the sensitivity of Positive (wild-type) + 1 ng the reaction of pGG55 (D133V) 7 ADV452 ADV453 100 fg of pGG55 Test the sensitivity of Positive (wild-type) + 1 ng the reaction pGG55 (D133V) 8 ADV452 ADV453 10 fg of pGG55 Test the sensitivity of Positive (wild-type) + the reaction pGG55 (D133V) 9 ADV452 ADV453 1 fg of pGG55 Test the sensitivity of Weakly (wild-type) + the reaction positive pGG55 (D133V) 10 ADV452 ADV453 0.1 fg of pGG55 Test the sensitivity of To be (wild-type) + the reaction determined pGG55 (D133V)

TABLE 4 Master PCR mix preparation. Reagent Quantity (μl) Water 206.25 Taq DNA Polymerase 10x reaction buffer 27.5 containing 15 mM MgCl₂ Deoxynucleotides (dNTPs) 10 mM each 5.5 Primers ADV452 (20 μM in water) 5.5 Primers ADV453 (20 μM in water) 5.5 pGG55 D133V (Lm-LLO-E7) plasmid (1 ng/μl) 11 Taq DNA Polymerase (5 U/μl) 2.75 Total 264

TABLE 5 PCR protocol for validation of the method to detect the presence of wild-type prfA sequence using primers ADV451, 452 and 453. Reagent PCR Water 18.75 μl  PCR Buffer 10x + MgCl₂ 15 mM 2.5 μl Deoxynucleotides mix (dATP, dCTP, dGTP and dTTP) 0.5 μl 10 mM each Primer ADV452 (20 μM) 0.5 μl Primer ADV453 (20 μM) 0.5 μl Taq DNA polymerase (5 U/μl) 0.25 μl  Template DNA (1 ng/μl) pGG55 D133V  1 μl Template DNA pGG55 WT (tubes 3 to 10)^(a)  1 μl Final volume per tube^(b)  25 μl ^(a)pGG55 WT (1 ng in tube 3; 100 pg in tube 4; 10 pg in tube 5; 1 pg in tube 6; 100 fg in tube 7; 10 fg in tube 8; 1 fg in tube 9; 0.1 fg in tube 10). ^(b)In tube 1, add 0.5 μl of water and 0.5 μl of primer ADV451 (20 μM stock); in tube 2 add 1 μl of water.

TABLE 6 PCR cycling conditions to detect the presence of wild- type prfA sequence using primers ADV451, 452 and 453. Step Temperature Time Number of cycles 1. 94° C. 2 minutes and 30 seconds 1 2. 94° C. 30 seconds 1 3. 53° C. 30 seconds 1 4. 72° C. 30 seconds 1 5. Repeat steps 2 to 4 12 6. 94° C. 30 seconds 1 7. 50° C. 30 seconds 1 8. 72° C. 30 seconds 1 9. Repeat steps 6 to 8 23 10. 72° C. 10 minutes 1

Sequencing:

Sequencing of the plasmids was done using the dideoxy sequencing method. The plasmids pGG55 D133V and pGG55 WT were mixed at different ratios (1:1, 1:10, 1; 100, 1:1,000 and 1:10,000). The total amount of plasmid in the mixture was kept constant (500 μg) and the plasmid containing the wild-type sequence was 10-fold serially diluted in relation to the D133V plasmid to determine the sensitivity of the method.

Results Example 6 Sequencing is not a Sensitive Method to Detect the Reversion of the D133V Mutation

To estimate the sensitivity of sequencing in detecting the wild-type prfA sequence, the pGG55 D133V and WT plasmids were mixed at the different ratios and sequenced. The results are shown in FIG. 12 and reveal that sequencing has a high specificity in discriminating the prfA D133V mutation (FIG. 12). On the other hand, the sensitivity is low and the maximum dilution of wild-type prfA pGG55 plasmid with a detectable peak in the sequence was 1 in 10 (FIG. 12). In conclusion, although sequencing is very specific, the sensitivity of the method is low and not appropriate to screen for the presence of rare events such as revertants of the prfA D133V mutation in Lm-LLO-E7 samples.

Example 7 Development of a Highly Specific and Sensitive PCR Method to Detect Reversion of the D133V Mutation

Given the low sensitivity of sequencing to detect rare events, it became imperative to develop a more sensitive method with similar specificity to detect reversion of the D133V mutation to wild-type. To achieve this goal, we designed a PCR-based method that specifically amplifies the wild-type sequence and is sensitive enough to detect at least 1 wild-type copy of prfA in 10,000,000 copies of the D133V mutated sequence. We designed 3 primers for this method: ADV451, ADV452 and ADV453 (Table 1). Both ADV451 and ADV452 are forward primers and differ in the last nucleotide at the 3′ position to discriminate the A→T (D133V) mutation at position 398 of the prfA gene. The ADV453 primer is the reverse primer located approximately 300 by downstream the annealing site of the ADV451 and ADV452 primers (FIG. 13). The expected PCR band obtained with the primers ADV451 or ADV452 and ADV453 is 326 bp. Under stringent conditions, the ADV451 primer should only amplify the pGG55 D133V plasmid, whereas the ADV452 would be specific to the wild-type prfA sequence.

Example 8 Specificity of the PCR Method

The reaction using the primer ADV451 was very specific and amplified the mutated D133V prfA sequence (lanes 1 to 3), but not the wild-type sequence (lanes 4 to 6). However, a very faint band can be detected in lane 4, when 5 μg of template DNA was used, but not with 1 μg (FIG. 14).

As shown in FIG. 15, the reaction with the ADV452 primer only amplified the wild-type prfA sequence (lanes 4, 5 and 6), and no bands were detected when the pGG55 carrying the D133V prfA mutation was used as a template (lanes 1, 2 and 3), even when using 5 μg of plasmid in the reaction (FIG. 16). In conclusion, the PCR reactions with primers ADV451 and ADV452 are very specific and able to discriminate the A

T (D133V) mutation at position 398 of the prfA gene in the pGG55 plasmid. Based on these results, we selected the amount of 1 μg as the standard amount of template DNA to be used in the reaction.

Example 9 Sensitivity of the PCR Method

The sensitivity of the reaction was tested using 1 μg of template DNA. For the plasmid carrying the wild-type prfA sequence, decreasing amounts of DNA (corresponding to 10-fold dilutions from 10⁻¹ to 10⁻⁷), were also included in the reaction to estimate the sensitivity. In these reactions only the primers ADV452 and ADV453 were used. In a PCR reaction with 30 cycles (10 cycles with annealing temperature of 53° C. and an additional 20 cycles with annealing temperature of 50° C.), the sensitivity of the method was 1 in 100,000 (data not shown). As shown in FIG. 5, increasing the number of PCR cycles to 37 improved the visual sensitivity of the method to 10⁻⁶ for the detection of D133V revertants, without significantly compromising the specificity. A clear band was visible at the 10⁻⁶ dilution, corresponding to a detection level of 1 copy of the wild-type sequence in a million of the D133V mutant, when 1 μg of plasmid was used as the initial amount of DNA. Only a very weak band can be visualized in lanes 1 and 9 after longer exposure, reassuring the robust specificity of the method. On the other hand, when starting with 5 μg of DNA, a band could be easily detected at the 10⁻⁷ dilution, increasing the sensitivity of the PCR. However, a similar band in intensity could also be detected with the pGG55 D133V plasmid, indicating the specificity limit of the method (FIG. 17). This band observed with the pGG55 D133V plasmid is likely due to non-specific amplification of the D133V mutation with primer ADV452 that can significantly accumulate with the increased number of cycles. These results indicate that the sensitivity limit for this method, without significantly compromising the specificity, is situated between 1 to 1,000,000 and 1 to 10,000,000.

Example 10 Recombinant Listeria Expressing a Fusion Protein of LLO to E7(Lm-LLO-E7)

This strain is approx. 4-5 logs more attenuated than the wild-type parent strain 10403S and secretes the fusion protein tLLO-E7. This immunotherapy is based on the backbone XFL7, which is derived from 10403S by the irreversible deletion in the virulence gene transcription activator prfA. PrfA regulates the transcription of several virulence genes such as Listeriolysin O (LLO), ActA, PlcA (phospholipase A), PlcB (phospholipase B) etc that are required for in vivo intracellular growth and survival of L. monocytogenes. The plasmid pGG55 is retained by the Lm-LLO-E7 in vitro by means of selection with ‘chloramphenicol’. However for in vivo retention of the plasmid by Lm-LLO-E7, it carries a copy of mutated prfA (D133V), which has been demonstrated to be less active than wild-type prfA in DNA binding and activating the transcription of virulence genes. We have observed that complementation with mutated prfA resulted in approx. 40 fold reduction in the amount of secreted LLO from Lm-LLO-E7 when compared to wild-type strain 10403S. This implicates that possibly the strain Lm-LLO-E7 exhibits a reduced expression of the virulence genes that are regulated by prfA such as actA, inlA, inlB, inlC, plcB etc. In Lm-LLO-E7, the complementation with mutated copy of prfA possibly causes a reduction in the expression of different virulence genes that are regulated by prfA resulting in overall attenuation of approx. 4-5 logs.

Example 11 Immunotherapy with Chemoradiation for Anal Cancer

Phase I-II Study

Eligibility Criteria

-   -   Newly diagnosed locally advanced anal cancer:     -   Stages: T≧4 cm or node +.     -   PS 0-1     -   Staging by CT/MRI or PET scan.     -   No significant cardiac or pulmonary disease. Adequate bone         marrow and renal function.

ADXS-HPV Treatment

-   -   ADXS-HPV administered at 1×10⁹cfu         -   IV Infusion of 500 ml over 30 minutes     -   Premedications for ADXS-HPV treatment:         -   Naprosyn 500 mg BID, Day −1, 0         -   Promethazine 25 mg PO, BID (pre-dose, 8 hours)     -   Post infusion antibiotics         -   Ampicillin 500 mg QID, Days 3-9

Treatment Schedule (See FIG. 23-24).

Patients with newly diagnosed anal cancer with a primary tumor >4 cm or lymph node involvement, without distant metastases, are eligible. All patients receive 2 courses of mitomycin, 5-FU with concurrent radiation (54 Gy in 30 fractions by intensity modulated radiation therapy [IMRT]). Patients receive 4 treatments of ADXS-HPV, 1×10⁹ colony forming units intravenously once approximately every 28 days. In treatment schedule #1 (FIG. 23), the first dose is given before chemoradiation and the 2-4^(th) doses are given every 28 days after completion of radiation. In treatment schedule #2 (FIG. 24), the second dose of ADXS-HPV is administered during chemoradiation.

Results

8 patients have been treated on the locally advanced anal cancer trial. Seven have had completed treatment and have no evidence of disease (NED) (Table 1). (Only the patient with the grade 5 unrelated cardiac event is not alive and with NED. —see Table 1)

Completed Treatment Patient Age Stage Standard Treatment Outcome 1 59 IIIB (T3N3) Yes CR, NED 2 50 IIIB (T2N2) Yes CR, NED 3 70 IIIA (T4N0) Yes CR, NED 4 55 IIIB (T3N3) Yes CR, NED 5 39 IIIA (T4N0) Yes CR, NED 6 71 II (4.5 cm, N0) Non tx related Gr5 Pending Cardiovascular 7 70 III T3NB Yes Pending 8 55 II (T3NO) Yes Pending

Toxicities

-   -   Rigors lasting for about 24 hours, often requiring meperidine,         are most common toxicity.     -   No evidence of bacterial infection.     -   No overlapping toxicities with chemoradiation (1 grade 3         neutropenia).

While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention. 

1. A method of treating an anal or vaginal tumor anal or vaginal cancer, or an anal or vaginal neoplasia in a human subject, the method comprising the step of administering to said subject a combination therapy comprising a chemo-radiation therapy and a recombinant Listeria strain, said Listeria strain comprising a recombinant nucleic acid, said nucleic acid comprising a first open reading frame encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a heterologous antigen or fragment thereof, thereby treating said anal or vaginal tumor anal or vaginal cancer, or an anal or vaginal neoplasia in said human subject.
 2. The method of claim 1, wherein said tumor, cancer, or neoplasia is an intraepithelial cancer or neoplasia.
 3. (canceled)
 4. The method of claim 1, wherein said chemo-radiation therapy is administered following a first administration of said recombinant Listeria strain.
 5. The method of claim 1, wherein said chemo-radiation therapy is administered prior to the administration of said recombinant Listeria strain.
 6. The method of claim 1, wherein said chemo-radiation therapy is administered following a first administration of said recombinant Listeria strain and prior to one to three booster administrations of said recombinant Listeria strain.
 7. The method of claim 1, wherein said chemo-radiation therapy is administered concurrently with said recombinant Listeria strain.
 8. The method of claim 1, wherein said method comprises administering four doses of said recombinant Listeria.
 9. The method of claim 8, wherein the first dose of said recombinant Listeria is administered prior to chemo-radiation therapy and the 2^(nd)-4^(th) doses are administered every 28 days after completion of radiation.
 10. The method of claim 8, wherein the first dose of said recombinant Listeria is administered before chemo-radiation therapy, wherein the second dose of said recombinant Listeria is administered during chemo-radiation therapy, and wherein the 3^(rd)-4^(th) doses are administered every 28 days following the completion of chemo-radiation therapy.
 11. The method of claim 1, wherein said chemo-radiation therapy comprises mitomycin and fluorouracil (5-FU) and radiation therapy.
 12. The method of claim 11, wherein said chemo-radiation therapy comprises administering 2 courses of mitomycin, 5-FU with concurrent radiation (54 Gy in 30 fractions by intensity modulated radiation therapy).
 13. The method of claim 11, wherein said radiation therapy lasts about 6 weeks.
 14. The method of claim 1, wherein said Listeria comprises a mutation or deletion in the endogenous prfA gene.
 15. The method of claim 14, wherein said recombinant nucleic acid further comprises a second open reading frame encoding a mutant prfA gene, wherein said mutant prfA gene complements the prfA genomic mutation or deletion, thereby inducing an immune response against said anal or vaginal tumor or anal or vaginal cancer.
 16. The method of claim 15, wherein said mutant prfA gene encodes a PrfA protein comprising a D133V mutation.
 17. (canceled)
 18. The method of claim 1, wherein said administering is intravenous or oral administering.
 19. The method of claim 1, wherein said N-terminal fragment of an LLO protein comprises SEQ ID NO:
 2. 20. The method of claim 1, wherein said recombinant Listeria strain is administered to said human subject at a dose of 1×10⁹-3.31×10¹⁰ organisms.
 21. The method of claim 1, wherein said recombinant Listeria strain is a recombinant Listeria monocytogenes strain.
 22. The method of claim 1, wherein said recombinant Listeria strain has been passaged through an animal host, prior to the step of administering.
 23. The method of claim 1, wherein said recombinant polypeptide is expressed by said recombinant Listeria strain.
 24. The method of claim 1, wherein said recombinant Listeria strain comprises a plasmid that encodes said recombinant polypeptide.
 25. (canceled)
 26. The method of claim 1, further comprising the step of inoculating said human subject with an immunogenic composition that comprises or directs expression of an HPV16 or HPV18 E6 or E7 antigen.
 27. The method of claim 1, wherein said recombinant Listeria strain has been stored in a frozen or lyophilized cell bank.
 28. A method for inducing an anti-tumor cytotoxic T cell response in a human subject, comprising the step of administering to said subject the combination therapy of claim
 1. 29. A method of treating a human subject against a tumor or cancer, comprising the step of administering to said subject the recombinant Listeria strain of claim
 1. 30. The method of claim 29, wherein said administering is intravenous or oral administering.
 31. The method of claim 28, wherein said immune response comprises increasing a level of interferon-gamma producing cells, increasing a level of TNF-alpha producing cells, or both.
 32. (canceled)
 33. The method of claim 28, wherein said immune response comprises an increase of tumor infiltration by T effector cells.
 34. The method of claim 33, wherein said T effector cells are CD8+T cells or CD4+T cells.
 35. The method of claim 28, wherein said immune response further comprises epitope spreading, induction of broad-based response to self-derived tumor antigens, or both.
 36. (canceled)
 37. The method of claim 28, wherein said immune response further comprises improvement of the overall balance of suppressor and effector immune cells in the tumor microenvironment or improvement in the systemic balance of suppressor and effector immunocytes. 38-150. (canceled)
 151. The method claim 12, wherein said radiation therapy lasts about 6 weeks.
 152. The method of claim 1, wherein said heterologous antigen is an HPV16 or HPV18 E6 or E7 antigen. 